Methods and compositions relating to anti-chi3l1 antibody reagents

ABSTRACT

Described herein are methods and compositions relating to anti-Chi3L1 antibodies, antibody reagents, and antigen-binding fragments thereof which display superior properties, e.g., high sensitivity, high specificity, high binding affinity, neutralization activity ex vivo and in vivo (e.g., blocks Chi3L1-induced MAPK and AKT signaling). Methods of treatment, e.g., of treating cancer, obesity, and/or asthma by administering the compounds described herein are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 365(c) and 35 U.S.C. §120 of International Application No. PCT/US18/012494 filed Jan. 5, 2018,which designates the U.S. and which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 62/442,513 filed Jan. 5,2017, the contents of which are incorporated herein by reference intheir entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. UH2 HL123876 awarded by the National Institutes of Health. The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 10, 2018, isnamed 2018-01-05_Seq_Listing_058040-088262-PCT.txt and is 27,672 bytesin size.

TECHNICAL FIELD

The technology described herein relates to antibodies and antibody-basedreagents that are specific for CHI3L1 and methods of using thosecompositions, e.g., to treat cancer or asthma.

BACKGROUND

The chitinase-like protein called chitinase 3-like-1 (Chi3l1; alsocalled Chi1l in mice and YKL-40 in man) has been implicated in asthmaand cancer, e.g., lung cancer. It has been demonstrated that the levelsof circulating Chi3l1 are increased in human asthma where they correlatewith disease severity. The levels of circulating YKL-40 are increased inmany malignancies including cancers of the prostate, colon, rectum,ovary, kidney, breast, glioblastomas and malignant melanoma. In thesediseases, the levels of YKL-40 frequently correlate directly withdisease progression and inversely with disease-free interval andsurvival.

SUMMARY

Described herein are the development and characterization of anti-CHI3L1antibodies demonstrated to have high specificity and the ability toblock CHI3L1 activity. These antibodies are further demonstrated to havetherapeutic activity in asthma and cancer models.

In one aspect of any of the embodiments, described herein is anantibody, antibody reagent, antigen-binding fragment thereof, orchimaeric antigen receptor (CAR), that specifically binds an CHI3L1polypeptide, said antibody reagent, antigen-binding portion thereof, orCAR comprising at least one heavy or light chain complementaritydetermining region (CDR) selected from the group consisting of:

-   -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or

a conservative substitution variant of one or more of (a)-(f).

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, or CAR comprises heavy chainCDRs having the amino acid sequences of SEQ ID NOs: 1-3 or aconservative substitution variant of such amino acid sequence. In someembodiments of any of the aspects, the antibody, antibody reagent,antigen-binding portion thereof, or CAR comprises light chain CDRshaving the amino acid sequences of SEQ ID NOs: 4-6 or a conservativesubstitution variant of such amino acid sequence. In some embodiments ofany of the aspects, the antibody, antibody reagent, antigen-bindingportion thereof, or CAR comprises light chain CDRs having the amino acidsequences of SEQ ID NOs: 4-6 and heavy chain CDRs having the amino acidsequences of SEQ ID NOs: 1-3 or a conservative substitution variant ofsuch amino acid sequence.

In one aspect of any of the embodiments, described herein is anantibody, antibody reagent, antigen-binding portion thereof, or CAR thatspecifically binds an CHI3L1 polypeptide, and can compete for binding ofCHI3L1 with an antibody comprising light chain CDRs having the aminoacid sequences of SEQ ID NOs: 4-6 and heavy chain CDRs having the aminoacid sequences of SEQ ID NOs: 1-3.

In some embodiments of any of the aspects, the antibody, antibodyreagent or antigen-binding fragment thereof binds to the epitope of SEQID NO: 13.

In one aspect of any of the embodiments, described herein is anantibody, antibody reagent, antigen-binding portion thereof, or CAR ofclaim 5 or 6, wherein the antibody, antibody reagent or antigen-bindingfragment thereof binds an CHI3L1 polypeptide at an eptitope selectedfrom SEQ ID NOs: 13-24.

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, or CAR further comprises aconservative substitution in a sequence not comprised by a CDR. In someembodiments of any of the aspects, the antibody, antibody reagent,antigen-binding portion thereof, or CAR is fully human or fullyhumanized. In some embodiments of any of the aspects, the antibody,antibody reagent, antigen-binding portion thereof, or CAR is fullyhumanized except for the CDR sequences.

In some embodiments of any of the aspects, the reagent or fragment isselected from the group consisting of: an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahumanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linkedFv, a scFv, a single domain antibody, a diabody, a multispecificantibody, a dual specific antibody, an anti-idiotypic antibody, and abispecific antibody.

In one aspect of any of the embodiments of any of the aspects, describedherein is a composition comprising the antibody, antibody reagent,antigen-binding portion thereof, or CAR as described herein, and achemotherapeutic agent. In some embodiments of any of the aspects,wherein the antibody, antibody reagent, or antigen-binding portionthereof is conjugated to the chemotherapeutic agent.

In one aspect of any of the embodiments, described herein is a nucleicacid sequence encoding the antibody, antibody reagent, antigen-bindingfragment thereof, or CAR as described herein, wherein at least one CDRis encoded by a nucleic acid sequence selected from SEQ ID NOs: 7-12.

In one aspect of any of the embodiments, described herein is a cellcomprising the antibody, antibody reagent, antigen-binding fragmentthereof, CAR or the nucleic acid sequence as described herein.

In one aspect of any of the embodiments, described herein is apharmaceutical composition comprising the antibody, antibody reagent,antigen-binding fragment thereof, CAR, composition, or cell as describedherein and a pharmaceutically acceptable carrier.

In one aspect of any of the embodiments, described herein is a solidsupport comprising the antibody, antibody reagent, antigen-bindingfragment thereof, or CAR as described herein. In some embodiments of anyof the aspects, the antibody, antibody reagent, antigen-binding fragmentthereof, or CAR is detectably labeled. In some embodiments of any of theaspects, the solid support comprises a particle, a bead, a polymer, or asubstrate.

In one aspect of any of the embodiments, described herein is a kit forthe detection of CHI3L1 polypeptide in a sample, the kit comprising atleast a first antibody, antibody reagent, antigen-binding fragmentthereof, or CAR as described herein, immobilized on a solid support andcomprising a detectable label.

In one aspect of any of the embodiments, described herein is amolecularcomplex comprising at least one antibody, antibody reagent,antigen-binding fragment thereof, or CAR as described herein bound to anCHI3L1 polypeptide.

In one aspect of any of the embodiments, described herein is a method oftreating cancer in a subject in need thereof, the method comprisingadministering the antibody, antibody reagent, antigen-binding fragmentthereof, CAR, composition, or cell as described herein to the subject.In some embodiments of any of the aspects, the cancer is primary cancer.In some embodiments of any of the aspects, the cancer is malignantcancer. In some embodiments of any of the aspects, the cancer isselected from the group consisting of: prostate cancer, colon cancer,rectal cancer, ovarian cancer, kidney cancer, breast cancer,glioblastoma, melanoma, malignant melanoma, and lung cancer.

In one aspect of any of the embodiments, described herein is a method oftreating asthma in a subject in need thereof, the method comprisingadministering the antibody, antibody reagent, antigen-binding fragmentthereof, CAR, composition, or cell as described herein to the subject.

In one aspect of any of the embodiments, described herein is a method oftreating obesity and/or preventing weight gain in a subject in needthereof, the method comprising administering the antibody, antibodyreagent, antigen-binding fragment thereof, CAR, composition, or cell asdescribed herein to the subject. In one aspect of any of theembodiments, described herein is a method of promoting weight loss in asubject in need thereof, the method comprising administering theantibody, antibody reagent, antigen-binding fragment thereof, CAR,composition, or cell as described herein to the subject.

In some embodiments of any of the aspects, the subject is a subjectdetermined to have an elevated level of CHI3L1. In some embodiments ofany of the aspects, the CHI3L1 is circulating CHI3L1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the characterization of the FRG monoclonal antibody(mAb). FIG. 1A demonstrates mAb analysis in Coomassie staining, Westernblot and Isotyping. FIG. 1B depicts FRG detection of Chi3l1 innon-denaturing and denaturing conditions. FIG. 1C depicts Sensitivityand specificity of FRG against recombinant (r) human and mouse Chi3l1detected by Western blot. FIG. 1D depicts FRG affinity and dose responsecurve evaluated by ELISA.

FIGS. 2A-2B demonstrate the neutralizing effects of FRG onChi3l1-stimulated signaling. FIG. 2A depicts effects on peritonealmacrophages*. FIG. 2B depicts effects on peritoneal macrophages—doseresponse*. * Thp1 cells, U937 cells, and AMJ2-C11 (mouse alveolarmacrophages cell line) showed similar pattern of inhibition and doseresponses on Chi3l1-stimulated Erk and Akt activation.

FIG. 3 depicts graphs of the effects of FRG on allergen-stimulated lunginflammation.

FIG. 4 depicts the effects of FRG on melanoma metastasis. Mice receivedB16-F10 cells by tail vein injection and were treated with control IGg2bantibody or FRG (200 μg/per mouse) every other day. The mice weresacrificed after 14 days and metastasis were evaluated.

FIGS. 5A-5B demonstrate the effects of FRG on primary lung cancer. FIG.5A depicts representative lungs from the KRAS/p53 mutant mice treatedwith control (Ctrl) igG or FRG mAb.

FIG. 5B depicts a representative histology of the lungs illustrated inFIG. 5A.

FIG. 6 depicts the location of selected epitopes including FRG in humanChi3l1.

FIG. 7 depicts the light chain CDR sequences of the FRG antibodydescribed herein. Figure discloses SEQ ID NOS 32-33, respectively, inorder of appearance.

FIG. 8 depicts the heavy chain CDR sequences of the FRG antibodydescribed herein. Figure discloses SEQ ID NOS 34-35, respectively, inorder of appearance.

FIG. 9 depicts a graph of WT mouse whole body weight changes after 14week HFD with & without antiChi3l1-FRG

FIG. 10 depicts a graph of absolute mouse body weight increases after 14week HFD with & without antiChi3l1-FRG

FIG. 11 depicts an image and a graph of body weight changes at 14th weekHFD with & without FRG

FIG. 12 depicts a graph of murine weight changes after 12 weeks on a HFDwith or without FRG antibody (200 μg/twice per week).

FIG. 13 depicts images of the effect of FRG antibody administration onabdominal fat accumulation after 12 weeks on a HFD with or without FRGantibody (200 μg/twice per week).

FIG. 14 depicts images of the effect of FRG antibody administration onthe indicated fat pads after 12 weeks on a HFD with or without FRGantibody (200 μg/twice per week).

FIG. 15 depicts a graph of murine serum glucose levels after 16 weeks ona HFD with or without FRG antibody at the indicated doses.

FIG. 16 depicts a graph of murine liver trigyceride levels after 16weeks on a HFD with or without FRG antibody at the indicated doses.

DETAILED DESCRIPTION

Described herein are antibodies, antibody reagents, antigen-bindingfragments thereof, or chamaeric antigen receptors (CARs) thatspecifically bind a CHI3L1 polypeptide. Such antibodies, antigen bindingportions thereof, etc., can permit, e.g., the diagnosis, prognosis,and/or treatment of cancer or asthma. In some embodiments, thetechnology described herein relates to chimeric antigen receptors (CARs)and CAR-T therapy for cancer or asthma. In some embodiments, thetechnology described herein relates to monoclonal antibody therapy forcancer or asthma. In some embodiments, the technology described hereinrelates to antibody-drug conjugates for the treatment of cancer orcancer.

Described herein are methods and compositions relating to anti-Chi3L1antibodies, antibody reagents, and antigen-binding fragments thereofwhich display superior properties, e.g., high sensitivity, highspecificity, high binding affinity, neutralization activity ex vivo andin vivo (e.g., blocks Chi3L1-induced MAPK and AKT signaling). Methods oftreatment, e.g., of treating cancer, obesity, and/or asthma byadministering the compounds described herein are also provided.

As used herein, “CHI3L1,” “chintinase-3-like protein 1,” or “YKL-40”refers to a ˜40 kDa glycoprotein secreted by at least macrophages,chondrocytes, neutrophils, synovial cells, and some cancer cells. CHI3L1does not have chitinase activity, is a Th2 promoting cytokine, has beenlinked to the AKT anti-apoptotic signaling pathway and induces themigration of astrocytes. The sequences of CHI3L1 expression products areknown for a number of species, e.g., human CHI3L1 (NCBI Gene ID No:1116) mRNA (SEQ ID NO: 31; NCBI Ref Seq: NM_001276.1 and SEQ ID NO: 26;NCBI Ref Seq: NM_001276.2) and polypeptide (SEQ ID NO: 27; NCBI Ref Seq:NP_001267.1 and SEQ ID NO: 28; NCBI Ref Seq: NP_001267.2).

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The term also refers to antibodies comprised of twoimmunoglobulin heavy chains and two immunoglobulin light chains as wellas a variety of forms including full length antibodies andantigen-binding portions thereof; including, for example, animmunoglobulin molecule, a monoclonal antibody, a chimeric antibody, aCDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, aFv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), adiabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding portion thereof, and/or bifunctional hybrid antibodies.

Each heavy chain is composed of a variable region of said heavy chain(abbreviated here as HCVR or VH) and a constant region of said heavychain. The heavy chain constant region consists of three domains CH1,CH2 and CH3. Each light chain is composed of a variable region of saidlight chain (abbreviated here as LCVR or VL) and a constant region ofsaid light chain. The light chain constant region consists of a CLdomain. The VH and VL regions may be further divided into hypervariableregions referred to as complementarity-determining regions (CDRs) andinterspersed with conserved regions referred to as framework regions(FR). Each VH and VL region thus consists of three CDRs and four FRswhich are arranged from the N terminus to the C terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure iswell known to those skilled in the art.

As used herein, the term “CDR” refers to the complementarity determiningregions within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and of the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The exact boundaries of these CDRs have been defined differentlyaccording to different systems. The system described by Kabat (Kabat etal., Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md. (1987) and (1991)) not only providesan unambiguous residue numbering system applicable to any variableregion of an antibody, but also provides precise residue boundariesdefining the three CDRs. These CDRs may be referred to as Kabat CDRs.Other boundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)) and Chothia (J. Mol. Biol. 196:901-917 (1987)and Nature 342:877-883 (1989)). Still other CDR boundary definitions maynot strictly follow one of the above systems, but will nonethelessoverlap with the Kabat CDRs, although they may be shortened orlengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughpreferred embodiments use Kabat defined CDRs. The CDR's identifiedherein, e.g., SEQ ID NOs 1-6 are identified by the Kabat system (see,e.g. FIGS. 7 and 8).

The term “antigen-binding portion” of an antibody refers to one or moreportions of an antibody as described herein, said portions) still havingthe binding affinities as defined above herein. Portions of a completeantibody have been shown to be able to carry out the antigen-bindingfunction of an antibody. In accordance with the term “antigen-bindingportion” of an antibody, examples of binding portions include (i) an Fabportion, i.e., a monovalent portion composed of the VL, VH, CL and CH1domains; (ii) an F(ab′)2 portion, i.e., a bivalent portion comprisingtwo Fab portions linked to one another in the hinge region via adisulfide bridge; (iii) an Fd portion composed of the VH and CH1domains; (iv) an Fv portion composed of the FL and VH domains of asingle arm of an antibody; and (v) a dAb portion consisting of a VHdomain or of VH, CH1, CH2, DH3, or VH, CH2, CH3 (dAbs, or single domainantibodies, comprising only VL domains have also been shown tospecifically bind to target eptiopes). Although the two domains of theFv portion, namely VL and VH, are encoded by separate genes, they mayfurther be linked to one another using a synthetic linker, e.g., apoly-G4S amino acid sequence (‘G4S’ disclosed as SEQ ID NO: 29), andrecombinant methods, making it possible to prepare them as a singleprotein chain in which the VL and VH regions combine in order to formmonovalent molecules (known as single chain Fv (ScFv)). The term“antigen-binding portion” of an antibody is also intended to comprisesuch single chain antibodies. Other forms of single chain antibodiessuch as “diabodies” are likewise included here. Diabodies are bivalent,bispecific antibodies in which VH and VL domains are expressed on asingle polypeptide chain, but using a linker which is too short for thetwo domains being able to combine on the same chain, thereby forcingsaid domains to pair with complementary domains of a different chain andto form two antigen-binding sites. An immunoglobulin constant domainrefers to a heavy or light chain constant domain. Human IgG heavy chainand light chain constant domain amino acid sequences are known in theart.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments, anantibody reagent can comprise a monoclonal antibody or a polypeptidecomprising an antigen-binding domain of a monoclonal antibody. Forexample, an antibody can include a heavy (H) chain variable region(abbreviated herein as VH), and a light (L) chain variable region(abbreviated herein as VL). In another example, an antibody includes twoheavy (H) chain variable regions and two light (L) chain variableregions. The term “antibody reagent” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab and sFabfragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domainantibodies (dAb) fragments as well as complete antibodies.

An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM(as well as subtypes and combinations thereof). Antibodies can be fromany source, including mouse, rabbit, pig, rat, and primate (human andnon-human primate) and primatized antibodies. Antibodies also includemidibodies, humanized antibodies, chimeric antibodies, and the like.

Furthermore, an antibody, antigen-binding portion thereof, or CAR asdescribed herein may be part of a larger immunoadhesion molecule formedby covalent or noncovalent association of said antibody or antibodyportion with one or more further proteins or peptides. Relevant to suchimmunoadhesion molecules are the use of the streptavidin core region inorder to prepare a tetrameric scFv molecule and the use of a cysteinresidue, a marker peptide and a C-terminal polyhistidinyl, e.g.,hexahistidinyl tag (‘hexahistidinyl tag’ disclosed as SEQ ID NO: 30) inorder to produce bivalent and biotinylated scFv molecules.

In some embodiments, the antibody, antibody reagent, antigen-bindingportion thereof, or CAR described herein can be an immunoglobulinmolecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, adisulfide linked Fv, a scFv, a single domain antibody, a diabody, amultispecific antibody, a dual specific antibody, an anti-idiotypicantibody, a bispecific antibody, and a functionally activeepitope-binding portion thereof.

In some embodiments, the antibody or antigen-binding portion thereof isa fully human antibody. In some embodiments, the antibody,antigen-binding portion thereof, is a humanized antibody or antibodyreagent. In some embodiments, the antibody, antigen-binding portionthereof, is a fully humanized antibody or antibody reagent. In someembodiments, the antibody or antigen-binding portion thereof, is achimeric antibody or antibody reagent. In some embodiments, theantibody, antigen-binding portion thereof, is a recombinant polypeptide.In some embodiments, the CAR comprises an extracellular domain thatbinds CHI3L1, wherein the extracellular domain comprises a humanized orchimeric antibody or antigen-binding portion thereof.

The term “human antibody” refers to antibodies whose variable andconstant regions correspond to or are derived from immunoglobulinsequences of the human germ line, as described, for example, by Kabat etal. (see Kabat, et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). However, the human antibodies can containamino acid residues not encoded by human germ line immunoglobulinsequences (for example mutations which have been introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs, and in particular in CDR3. Recombinant humanantibodies as described herein have variable regions and may alsocontain constant regions derived from immunoglobulin sequences of thehuman germ line (see Kabat, E. A., et al. (1991) Sequences of Proteinsof Immunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). According to particularembodiments, however, such recombinant human antibodies are subjected toin-vitro mutagenesis (or to a somatic in-vivo mutagenesis, if an animalis used which is transgenic due to human Ig sequences) so that the aminoacid sequences of the VH and VL regions of the recombinant antibodiesare sequences which although related to or derived from VH and VLsequences of the human germ line, do not naturally exist in vivo withinthe human antibody germ line repertoire. According to particularembodiments, recombinant antibodies of this kind are the result ofselective mutagenesis or back mutation or of both. Preferably,mutagenesis leads to an affinity to the target which is greater, and/oran affinity to non-target structures which is smaller than that of theparent antibody. Generating a humanized antibody from the sequences andinformation provided herein can be practiced by those of ordinary skillin the art without undue experimentation. In one approach, there arefour general steps employed to humanize a monoclonal antibody, see,e.g., U.S. Pat. No. 5,585,089; U.S. Pat. No. 6,835,823; U.S. Pat. No.6,824,989. These are: (1) determining the nucleotide and predicted aminoacid sequence of the starting antibody light and heavy variable domains;(2) designing the humanized antibody, i.e., deciding which antibodyframework region to use during the humanizing process; (3) the actualhumanizing methodologies/techniques; and (4) the transfection andexpression of the humanized antibody.

Usually the CDR regions in humanized antibodies and human antibodyvariants are substantially identical, and more usually, identical to thecorresponding CDR regions in the mouse or human antibody from which theywere derived. In some embodiments, it is possible to make one or moreconservative amino acid substitutions of CDR residues withoutappreciably affecting the binding affinity of the resulting humanizedimmunoglobulin or human antibody variant. In some embodiments,substitutions of CDR regions can enhance binding affinity.

The term “chimeric antibody” refers to antibodies which containsequences for the variable region of the heavy and light chains from onespecies and constant region sequences from another species, such asantibodies having murine heavy and light chain variable regions linkedto human constant regions. Humanized antibodies have variable regionframework residues substantially from a human antibody (termed anacceptor antibody) and complementarity determining regions substantiallyfrom a non-human antibody, e.g., a mouse-antibody, (referred to as thedonor immunoglobulin). The constant region(s), if present, are alsosubstantially or entirely from a human immunoglobulin. The humanvariable domains are usually chosen from human antibodies whoseframework sequences exhibit a high degree of sequence identity with the(murine) variable region domains from which the CDRs were derived. Theheavy and light chain variable region framework residues can besubstantially similar to a region of the same or different humanantibody sequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies.

In addition, techniques developed for the production of “chimericantibodies” by splicing genes from a mouse, or other species, antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.The variable segments of chimeric antibodies are typically linked to atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. Human constant region DNA sequences canbe isolated in accordance with well-known procedures from a variety ofhuman cells, such as immortalized B-cells. The antibody can contain bothlight chain and heavy chain constant regions. The heavy chain constantregion can include CH1, hinge, CH2, CH3, and, sometimes, CH4 regions.For therapeutic purposes, the CH2 domain can be deleted or omitted.

Additionally, and as described herein, a recombinant humanized antibodycan be further optimized to decrease potential immunogenicity, whilemaintaining functional activity, for therapy in humans. In this regard,functional activity means a polypeptide capable of displaying one ormore known functional activities associated with a recombinant antibody,antigen-binding portion thereof, or CAR as described herein. Suchfunctional activities include binding to cancer cells and/or anti-canceractivity. Additionally, a polypeptide having functional activity meansthe polypeptide exhibits activity similar, but not necessarily identicalto, an activity of a reference antibody, antigen-binding portionthereof, or CAR as described herein, including mature forms, as measuredin a particular assay, such as, for example, a biological assay, with orwithout dose dependency. In the case where dose dependency does exist,it need not be identical to that of the reference antibody,antigen-binding portion thereof, or CAR, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thereference antibody, antigen-binding portion thereof, or CAR as describedherein (i.e., the candidate polypeptide will exhibit greater activity,or not more than about 25-fold less, about 10-fold less, or about 3-foldless activity relative to the antibodies, antigen-binding portions,and/or CARS described herein).

In some embodiments, the antibody reagents (e.g., antibodies or CARs)described herein are not naturally-occurring biomolecules. For example,a murine antibody raised against an antigen of human origin would notoccur in nature absent human intervention and manipulation, e.g.,manufacturing steps carried out by a human. Chimeric antibodies are alsonot naturally-occurring biomolecules, e.g., in that they comprisesequences obtained from multiple species and assembled into arecombinant molecule. In certain particular embodiments, the humanantibody reagents described herein are not naturally-occurringbiomolecules, e.g., fully human antibodies directed against a humanantigen would be subject to negative selection in nature and are notnaturally found in the human body.

In some embodiments, the antibody, antibody reagent, antigen-bindingportion thereof, and/or CAR is an isolated polypeptide. In someembodiments, the antibody, antibody reagent, antigen-binding portionthereof, and/or CAR is a purified polypeptide. In some embodiments, theantibody, antibody reagent, antigen-binding portion thereof, and/or CARis an engineered polypeptide.

In one aspect of any of the embodiments, described herein is anantibody, antigen-binding fragment thereof, antigen reagent or chimaericantigen receptor (CAR), that specifically binds a CHI3L1 polypeptide. Insome embodiments of any of the aspects, the antibody, antigen-bindingfragment thereof, antigen reagent or CAR comprises at least one heavy orlight chain complementarity determining region (CDR) selected from thegroup consisting of:

-   -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or        a conservative substitution variant of one or more of (a)-(f).        In some embodiments of any of the aspects, the antibody,        antigen-binding fragment thereof, antigen reagent or CAR        comprises at least one heavy or light chain complementarity        determining region (CDR) selected from the group consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3. In some embodiments of any of the aspects, the antibody,        antibody reagent, antigen-binding portion thereof, or CAR that        specifically binds an CHI3L1 polypeptide binds specifically to        an epitope selected from SEQ ID NOs: 13-24. In some embodiments        of any of the aspects, the antibody, antibody reagent,        antigen-binding portion thereof, or CAR that specifically binds        an CHI3L1 polypeptide binds specifically to the epitope of SEQ        ID NO: 13.

In some embodiments of any of the aspects, the antibody, antigen-bindingfragment thereof, antigen reagent or CAR comprises heavy chain CDRshaving the amino acid sequences of SEQ ID NOs: 1-3. In some embodimentsof any of the aspects, the antibody, antigen-binding fragment thereof,antigen reagent or CAR comprises heavy chain CDRs having the amino acidsequences of SEQ ID NOs: 1-3 or a conservative substitution variant ofsuch amino acid sequence.

In some embodiments of any of the aspects, the antibody, antigen-bindingfragment thereof, antigen reagent or CAR comprises light chain CDRshaving the amino acid sequences of SEQ ID NOs: 4-6. In some embodimentsof any of the aspects, the antibody, antigen-binding fragment thereof,antigen reagent or CAR comprises light chain CDRs having the amino acidsequences of SEQ ID NOs: 4-6 or a conservative substitution variant ofsuch amino acid sequence.

In some embodiments of any of the aspects, the antibody, antigen-bindingfragment thereof, antigen reagent or CAR comprises the heavy or lightchain complementarity determining region (CDR)s as follows:

-   -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3.        In some embodiments of any of the aspects, the antibody,        antigen-binding fragment thereof, antigen reagent or CAR        comprises the heavy or light chain complementarity determining        region (CDR)s as follows:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or a conservative subsitutition variant of the amino acid        sequence of any of (a)-(f). In some embodiments of any of the        aspects, the antibody, antibody reagent, antigen-binding portion        thereof, or CAR that specifically binds an CHI3L1 polypeptide        binds specifically to an epitope selected from SEQ ID NOs:        13-24. In some embodiments of any of the aspects, the antibody,        antibody reagent, antigen-binding portion thereof, or CAR that        specifically binds an CHI3L1 polypeptide binds specifically to        the epitope of SEQ ID NO: 13.

In some embodiments, the antibody, antibody reagent, antigen-bindingportion thereof, or CAR can comprise one or more CDRs (e.g., one CDR,two CDRs, three CDRs, four CDRs, five CDRs, or six CDRs) having thesequence of a CDR selected from SEQ ID NOs: 1-6. In some embodiments,the antibody, antibody reagent, antigen-binding portion thereof, or CARcan comprise CDRs having the sequence of the CDRs of SEQ ID NOs: 1-6.

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, or CAR can comprise a heavychain sequence having the amino acid sequence of SEQ ID NO: 36 and/or alight chain sequence having the amino acid sequence of SEQ ID NO: 38.

In one aspect of any of the embodiments, described herein is anantibody, antibody reagent, antigen-binding portion thereof, or CAR thatspecifically binds an CHI3L1 polypeptide, and can compete for binding ofCHI3L1 with an antibody comprising light chain CDRs having the aminoacid sequences of SEQ ID NOs: 4-6 and heavy chain CDRs having the aminoacid sequences of SEQ ID NOs: 1-3. In some embodiments of any of theaspects, the antibody, antibody reagent, antigen-binding portionthereof, or CAR that specifically binds an CHI3L1 polypeptide bindsspecifically to an epitope selected from SEQ ID NOs: 13-24. In someembodiments of any of the aspects, the antibody, antibody reagent,antigen-binding portion thereof, or CAR that specifically binds anCHI3L1 polypeptide binds specifically to the epitope of SEQ ID NO: 13.

In some embodiments, the antibody, antibody reagent, antigen-bindingportion thereof, and/or CAR as described herein can be a variant of asequence described herein, e.g., a conservative substitution variant ofan antibody polypeptide. In some embodiments, the variant is aconservatively modified variant. Conservative substitution variants canbe obtained by mutations of native nucleotide sequences, for example. A“variant,” as referred to herein, is a polypeptide substantiallyhomologous to a native or reference polypeptide, but which has an aminoacid sequence different from that of the native or reference polypeptidebecause of one or a plurality of deletions, insertions or substitutions.Variant polypeptide-encoding DNA sequences encompass sequences thatcomprise one or more additions, deletions, or substitutions ofnucleotides when compared to a native or reference DNA sequence, butthat encode a variant protein or portion thereof that retains activity,e.g., antigen-specific binding activity for the relevant targetpolypeptide, e.g., CHI3L1. A wide variety of PCR-based site-specificmutagenesis approaches are also known in the art and can be applied bythe ordinarily skilled artisan.

One of skill will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide, or protein sequencewhich alters a single amino acid or a small percentage of amino acids inthe encoded sequence is a “conservatively modified variant” where thealteration results in the substitution of an amino acid with achemically similar amino acid and retain the ability to specificallybind the target antigen (e.g., CHI3L1). Such conservatively modifiedvariants are in addition to and do not exclude polymorphic variants,interspecies homologs, and alleles consistent with the disclosure.

Examples of substitution variants include conservative substitution ofamino acids, e.g., in a VH or VL, domain, that do not alter the sequenceof a CDR. A conservative substitution in a sequence not comprised by aCDR can be a substitution relative to a wild-type or naturally-occurringsequence, e.g., human or murine framework and/or constant regions of anantibody sequence. In some embodiments, a conservatively modifiedvariant of an antibody reagent can comprise alterations other than inthe CDRs, e.g., a conservatively modified variant of an antibody,antibody reagent, antigen-binding portion thereof, or CAR can compriseCDRs having the sequence of one or more of SEQ ID NOs 1-6. In someembodiments, a conservatively modified variant of an antibody, antibodyreagent, antigen-binding portion thereof, or CAR can comprise CDRshaving the sequences of SEQ ID NOs: 1-6.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity, e.g.,antigen-binding activity and specificity of a native or referencepolypeptide is retained.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues can be divided intogroups based on common side-chain properties: (1) hydrophobic:Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser,Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5)residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp,Tyr, Phe. Non-conservative substitutions will entail exchanging a memberof one of these classes for another class. Particular conservativesubstitutions include, for example; Ala into Gly or into Ser; Arg intoLys; Asn into Gln or into H is; Asp into Glu; Cys into Ser; Gln intoAsn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln;Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, intoGln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, intoLeu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;and/or Phe into Val, into Ile or into Leu.

A variant amino acid or DNA sequence preferably is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or more, identicalto a native or reference sequence. The degree of homology (percentidentity) between a native and a mutant sequence can be determined, forexample, by comparing the two sequences using freely available computerprograms commonly employed for this purpose on the world wide web (e.g.,BLASTp or BLASTn with default settings).

Alterations of the native amino acid sequence can be accomplished by anyof a number of techniques known to one of skill in the art. Mutationscan be introduced, for example, at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered nucleotide sequencehaving particular codons altered according to the substitution,deletion, or insertion required.

Any cysteine residue not involved in maintaining the proper conformationof the polypeptide also can be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) can be added to thepolypeptide to improve its stability or facilitate oligomerization.

In particular embodiments wherein an antibody, antigen-binding portionthereof, or CAR as described herein comprises at least one CDR which isnot identical to the sequence of SEQ ID NOs: 1-6, the amino acidsequence of that at least one CDR can be selected by methods well knownto one of skill in the art. For example, Fujii, 2004, “Antibody affinitymaturation by random mutagenesis” in Methods in Molecular Biology:Antibody Engineering 248: 345-349 (incorporated by reference herein inits entirety), particularly at FIG. 2 and Section 3.3, describes methodsof generating a library for any CDR of interest. This allows one ofordinary skill in the art to identify alternative CDRs, includingconservative substitution variants of the specific CDR sequencesdescribed herein, which, when present in an antibody or antigen-bindingportion thereof as described herein, will result in an antigen orantigen-binding portion thereof which will bind a cancer cell surfaceantigen. The method described in Fujii et al. also permits one ofordinary skill in the art to screen for a light chain sequence whichwill give the desired binding behavior when combined with a known heavychain fragment and vice versa.

In some embodiments, a CAR comprises an extracellular domain comprisingan anti-CHI3L1 antibody or antigen-binding portion thereof that bindsone or more epitopes of a CHI3L1 polypeptide; a transmembrane domain,one or more intracellular co-stimulatory signaling domains, and aprimary signaling domain. Exemplary anti-CHI3L1 antibodies andantigen-binding portions thereof, as well as exemplary epitopes, aredescribed elsewhere herein

As used herein, “chimeric antigen receptor” or “CAR” refers to anartificially constructed hybrid polypeptide comprising anantigen-binding domain (e.g., an antigen-binding portion of an antibody(e.g., a scFV)), a transmembrane domain, and a T-cell signaling and/orT-cell activation domain. CARS have the ability to redirect T-cellspecificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givesT-cells expressing CARs the ability to recognize an antigen independentof antigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T-cells, CARS advantageously do not dimerizewith endogenous T-cell receptor (TCR) alpha and beta chains. Mostcommonly, the CAR's extracellular binding domain is composed of a singlechain variable fragment (scFv) derived from fusing the variable heavyand light regions of a murine or humanized monoclonal antibody.Alternatively, scFvs may be used that are derived from Fab's (instead offrom an antibody, e.g., obtained from Fab libraries), in variousembodiments, this scFv is fused to a transmembrane domain and then to anintracellular signaling domain. “First-generation” CARs include thosethat solely provide CD3zeta (CD3ζ) signals upon antigen binding,“Second-generation” CARS include those that provide both costimulation(e.g., CD28 or CD 137) and activation (CD3ζ). “Third-generation” CARSinclude those that provide multiple costimulatory (e.g., CD28 and CD137) domains and activation domains (e.g., CD3ζ). In variousembodiments, the CAR is selected to have high affinity or avidity forthe antigen. Further discussion of CARs can be found, e.g., in Maus etal. Blood 2014 123:2624-35; Reardon et al. Neuro-Oncology 201416:1441-1458; Hoyos et al. Haematologica 2012 97:1622; Byrd et al. JClin Oncol 2014 32:3039-47; Maher et al. Cancer Res 2009 69:4559-4562;and Tamada et al. Clin Cancer Res 2012 18:6436-6445; each of which isincorporated by reference herein in its entirety.

In some embodiments of any of the aspects, a CAR comprises anextracellular binding domain that comprises a humanized CHI3L1-specificbinding domain; a transmembrane domain; one or more intracellularco-stimulatory signaling domains; and a primary signaling domain. Asused herein, the terms, “binding domain,” “extracellular domain,”“extracellular binding domain,” “antigen-specific binding domain,” and“extracellular antigen specific binding domain,” are usedinterchangeably and provide a CAR with the ability to specifically bindto the target antigen of interest, e.g., CHI3L1. The binding domain maybe derived either from a natural, synthetic, semi-synthetic, orrecombinant source.

In some embodiments, the CARs contemplated herein may comprise linkerresidues between the various domains, e.g., added for appropriatespacing and conformation of the molecule. In particular embodiments thelinker is a variable region linking sequence. A “variable region linkingsequence,” is an amino acid sequence that connects the VH and VL domainsand provides a spacer function compatible with interaction of the twosub-binding domains so that the resulting polypeptide retains a specificbinding affinity to the same target molecule as an antibody thatcomprises the same light and heavy chain variable regions. CARscontemplated herein, can comprise one, two, three, four, or five or morelinkers. In particular embodiments, the length of a linker is about 1 toabout 25 amino acids, about 5 to about 20 amino acids, or about 10 toabout 20 amino acids, or any intervening length of amino acids. In someembodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acidslong.

In particular embodiments, the binding domain of the CAR is followed byone or more “spacer domains,” which refers to the region that moves theantigen binding domain away from the effector cell surface to enableproper cell/cell contact, antigen binding and activation. The hingedomain may be derived either from a natural, synthetic, semi-synthetic,or recombinant source. In certain embodiments, a spacer domain is aportion of an immunoglobulin, including, but not limited to, one or moreheavy chain constant regions, e.g., CH2 and CH3. The spacer domain caninclude the amino acid sequence of a naturally occurring immunoglobulinhinge region or an altered immunoglobulin hinge region.

The binding domain of the CAR is generally followed by one or more“hinge domains,” which plays a role in positioning the antigen bindingdomain away from the effector cell surface to enable proper cell/cellcontact, antigen binding and activation. A CAR generally comprises oneor more hinge domains between the binding domain and the transmembranedomain (TM). The hinge domain may be derived either from a natural,synthetic, semi-synthetic, or recombinant source. The hinge domain caninclude the amino acid sequence of a naturally occurring immunoglobulinhinge region or an altered immunoglobulin hinge region. Illustrativehinge domains suitable for use in the CARS described herein include thehinge region derived from the extracellular regions of type 1 membraneproteins such as CD8a, CD4, CD28 and CD7, which may be wild-type hingeregions from these molecules or may be altered. In another embodiment,the hinge domain comprises a CD8a hinge region.

The “transmembrane domain” is the portion of the CAR that fuses theextracellular binding portion and intracellular signaling domain andanchors the CAR to the plasma membrane of the immune effector cell. TheTM domain may be derived either from a natural, synthetic,semi-synthetic, or recombinant source. The TM domain may be derived from(i.e., comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD3c, CD3, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137,CD152, CD 154, and PD1.

In some embodiments, CARs contemplated herein comprise an intracellularsignaling domain. An “intracellular signaling domain,” refers to thepart of a CAR that participates in transducing the message of effectiveCAR binding to a target antigen into the interior of the immune effectorcell to elicit effector cell function, e.g., activation, cytokineproduction, proliferation and cytotoxic activity, including the releaseof cytotoxic factors to the CAR-bound target cell, or other cellularresponses elicited with antigen binding to the extracellular CAR domain.In some embodiments, a CAR contemplated herein comprises anintracellular signaling domain that comprises one or more“co-stimulatory signaling domain” and a “primary signaling domain.”

Primary signaling domains regulate primary activation of the TCR complexeither in a stimulatory way, or in an inhibitory way. Primary signalingdomains that act in a stimulatory manner may contain signaling motifswhich are known as immunoreceptor tyrosine-based activation motifs orITAMs. Illustrative examples of ITAM containing primary signalingdomains that are of particular use in the invention include thosederived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a,CD79b, and CD66d.

As used herein, the term, “co-stimulatory signaling domain,” or“co-stimulatory domain”, refers to an intracellular signaling domain ofa co-stimulatory molecule. Co-stimulatory molecules are cell surfacemolecules other than antigen receptors or Fc receptors that provide asecond signal required for efficient activation and function of Tlymphocytes upon binding to antigen. Illustrative examples of suchco-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30,CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1),CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1),CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In oneembodiment, a CAR comprises one or more co-stimulatory signaling domainsselected from the group consisting of CD28, CD137, and CD134, and a CD3primary signaling domain.

In some embodiments, an antibody-drug conjugate is provided. Inparticular embodiments, an antibody-drug conjugate comprises anantibody, antibody reagent, or antigen-binding portion thereof asdescribed herein. The drug can be, e.g., a chemotherapeutic molecule asdescribed elsewhere herein. In some embodiments, the antibody-drugconjugate comprises a chemotherapeutic agent directly conjugated and/orbound to an antibody or antigen-binding portion thereof. In someembodiments, binding can be non-covalent, e.g., by hydrogen bonding,electrostatic, or van der Waals interactions; however, binding may alsobe covalent. By “conjugated” is meant the covalent linkage of at leasttwo molecules. In some embodiments, the composition can be anantibody-drug conjugate.

In some embodiments, an antibody, antibody reagent, or antigen-bindingportion thereof can be bound to and/or conjugated to multiplechemotherapeutic molecules. In some embodiments, an antibody-drugconjugate can be bound to and/or conjugated to multiple chemotherapeuticmolecules. In some embodiments, the ratio of a given chemotherapeuticmolecule to an antibody or antigen-binding portion thereof can be fromabout 1:1 to about 1,000:1, e.g., a single antibody reagent molecule canbe linked to, conjugated to, etc. from about 1 to about 1,000 individualchemotherapeutic molecules.

In some embodiments, an antibody, or antigen-binding portion thereof,and the chemotherapeutic agent can be present in a scaffold material.Scaffold materials suitable for use in therapeutic compositions areknown in the art and can include, but are not limited to, ananoparticle; a matrix; a hydrogel; and a biomaterial, biocompatible,and/or biodegradable scaffold material. As used herein, the term“nanoparticle” refers to particles that are on the order of about 10⁻⁹or one to several billionths of a meter. The term “nanoparticle”includes nanospheres; nanorods; nanoshells; and nanoprisms; thesenanoparticles may be part of a nanonetwork.

The term “nanoparticles” also encompasses liposomes and lipid particleshaving the size of a nanoparticle. As used herein, the term “matrix”refers to a 3-dimensional structure comprising the components of acomposition described herein (e.g., an antibody or antigen-bindingportion thereof). Non-limiting examples of matrix structures includefoams; hydrogels; electrospun fibers; gels; fiber mats; sponges;3-dimensional scaffolds; non-woven mats; woven materials; knitmaterials; fiber bundles; and fibers and other material formats (See,e.g., Rockwood et al. Nature Protocols 2011 6:1612-1631 and US PatentPublications 2011/0167602; 2011/0009960; 2012/0296352; and U.S. Pat. No.8,172,901; each of which is incorporated by reference herein in itsentirety). The structure of the matrix can be selected by one of skillin the art depending upon the intended application of the composition,e.g., electrospun matrices can have greater surface area than foams.

In some embodiments, the scaffold is a hydrogel. As used herein, theterm “hydrogel” refers to a three-dimensional polymeric structure thatis insoluble in water but which is capable of absorbing and retaininglarge quantities of water to form a stable, often soft and pliable,structure. In some embodiments, water can penetrate in between thepolymer chains of the polymer network, subsequently causing swelling andthe formation of a hydrogel. In general, hydrogels are superabsorbent.Hydrogels have many desirable properties for biomedical applications.For example, they can be made nontoxic and compatible with tissue, andthey are highly permeable to water, ions, and small molecules. Hydrogelsare super-absorbent (they can contain over 99% water) and can becomprised of natural (e.g., silk) or synthetic polymers, e.g., PEG.

As used herein, “biomaterial” refers to a material that is biocompatibleand biodegradable. As used herein, the term “biocompatible” refers tosubstances that are not toxic to cells. In some embodiments, a substanceis considered to be “biocompatible” if its addition to cells in vitroresults in less than or equal to approximately 20% cell death. In someembodiments, a substance is considered to be “biocompatible” if itsaddition to cells in vivo does not induce inflammation and/or otheradverse effects in vivo. As used herein, the term “biodegradable” refersto substances that are degraded under physiological conditions. In someembodiments, a biodegradable substance is a substance that is brokendown by cellular machinery. In some embodiments, a biodegradablesubstance is a substance that is broken down by chemical processes.

In some embodiments, the technology described herein relates to anucleic acid encoding an antibody, antibody reagent, antigen-bindingportion thereof, or CAR as described herein. In some embodiments, thenucleic acid is a cDNA. In some embodiments, the one or more portions ofnucleic acid encoding CDR(s) comprises a sequence selected from SEQ IDNOs: 7-12. In some embodiments, the nucleic acid can comprise SEQ ID NO:37 and/or SEQ ID NO: 39.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to a polymeric molecule incorporating units of ribonucleic acid,deoxyribonucleic acid or an analog thereof. The nucleic acid can beeither single-stranded or double-stranded. A single-stranded nucleicacid can be one strand nucleic acid of a denatured double-stranded DNA.In some embodiments, the nucleic acid can be a cDNA, e.g., a nucleicacid lacking introns.

Nucleic acid molecules encoding amino acid sequence variants ofantibodies are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the antibody. A nucleic acid sequence encodingat least one antibody, portion or polypeptide as described herein can berecombined with vector DNA in accordance with conventional techniques,including blunt-ended or staggered-ended termini for ligation,restriction enzyme digestion to provide appropriate termini, filling inof cohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and ligation with appropriate ligases. Techniquesfor such manipulations can be used to construct nucleic acid sequenceswhich encode a monoclonal antibody molecule, antibody reagent, antigenbinding region thereof, or CAR.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression as peptides orantibody portions in recoverable amounts. The precise nature of theregulatory regions needed for gene expression may vary from organism toorganism, as is well known in the analogous art.

In some embodiments, a nucleic acid encoding an antibody, antibodyreagent, antigen-binding portion thereof, or CAR as described herein iscomprised by a vector. In some of the aspects described herein, anucleic acid sequence encoding an antibody, antibody reagent,antigen-binding portion thereof, or CAR as described herein, or anymodule thereof, is operably linked to a vector. The term “vector”, asused herein, refers to a nucleic acid construct designed for delivery toa host cell or for transfer between different host cells. As usedherein, a vector can be viral or non-viral. The term “vector”encompasses any genetic element that is capable of replication whenassociated with the proper control elements and that can transfer genesequences to cells. A vector can include, but is not limited to, acloning vector, an expression vector, a plasmid, phage, transposon,cosmid, chromosome, virus, virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g., 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the nucleic acid encoding an antibody, antigen-bindingportion thereof, or CAR as described herein in place of non-essentialviral genes. The vector and/or particle may be utilized for the purposeof transferring any nucleic acids into cells either in vitro or in vivo.Numerous forms of viral vectors are known in the art.

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments, be combined with other suitable compositions andtherapies. In some embodiments, the vector is episomal. The use of asuitable episomal vector provides a means of maintaining the nucleotideof interest in the subject in high copy number extra chromosomal DNAthereby eliminating potential effects of chromosomal integration.

In one aspect of any of the embodiments, described herein is a cellcomprising an antibody, antibody reagent, antigen-binding portionthereof, or CAR as described herein, or a nucleic acid encoding such anantibody, antibody reagent, antigen-binding portion thereof, or CAR.

The expression of an antibody, antibody reagent, antigen-binding portionthereof, or CAR as described herein can occur in either prokaryotic oreukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts,including yeast, insects, fungi, bird and mammalian cells either invivo, or in situ, or host cells of mammalian, insect, bird or yeastorigin. The mammalian cell or tissue can be of human, primate, hamster,rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but anyother mammalian cell may be used. Further, by use of, for example, theyeast ubiquitin hydrolase system, in vivo synthesis ofubiquitin-transmembrane polypeptide fusion proteins can be accomplished.The fusion proteins so produced can be processed in vivo or purified andprocessed in vitro, allowing synthesis of an antibody or portion thereofas described herein with a specified amino terminus sequence. Moreover,problems associated with retention of initiation codon-derivedmethionine residues in direct yeast (or bacterial) expression maybeavoided. Any of a series of yeast gene expression systems incorporatingpromoter and termination elements from the actively expressed genescoding for glycolytic enzymes produced in large quantities when yeastare grown in mediums rich in glucose can be utilized to obtainrecombinant antibodies or antigen-binding portions thereof as describedherein. Known glycolytic genes can also provide very efficienttranscriptional control signals. For example, the promoter andterminator signals of the phosphoglycerate kinase gene can be utilized.

Production of antibodies or antigen-binding portions thereof asdescribed herein in insects can be achieved. For example, by infectingthe insect host with a baculovirus engineered to express a transmembranepolypeptide by methods known to those of ordinary skill in the art.

In some embodiments, the introduced nucleotide sequence is incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors can be employed forthis purpose and are known and available to those or ordinary skill inthe art. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.

Example prokaryotic vectors known in the art include plasmids such asthose capable of replication in E. coli., for example. Other geneexpression elements useful for the expression of cDNA encodingantibodies, antigen-binding portions thereof, or CARS include, but arenot limited to (a) viral transcription promoters and their enhancerelements, such as the SV40 early promoter, Rous sarcoma virus LTR, andMoloney murine leukemia virus; (b) splice regions and polyadenylationsites such as those derived from the SV40 late region, and (c)polyadenylation sites such as in SV40. Immunoglobulin cDNA genes can beexpressed, e.g., using as expression elements the SV40 early promoterand its enhancer, the mouse immunoglobulin H chain promoter enhancers,SV40 late region mRNA splicing, rabbit S-globin intervening sequence,immunoglobulin and rabbit S-globin polyadenylation sites, and SV40polyadenylation elements.

For immunoglobulin genes comprised of part cDNA, part genomic DNA, thetranscriptional promoter can be human cytomegalovirus, the promoterenhancers can be cytomegalovirus and mouse/human immunoglobulin, andmRNA splicing and polyadenylation regions can be the native chromosomalimmunoglobulin sequences.

In some embodiments, for expression of cDNA genes in rodent cells, thetranscriptional promoter is a viral LTR sequence, the transcriptionalpromoter enhancers are either or both the mouse immunoglobulin heavychain enhancer and the viral LTR enhancer, the splice region contains anintron of greater than 31 bp, and the polyadenylation and transcriptiontermination regions are derived from the native chromosomal sequencecorresponding to the immunoglobulin chain being synthesized. In otherembodiments, cDNA sequences encoding other proteins are combined withthe above-recited expression elements to achieve expression of theproteins in mammalian cells.

A gene is assembled in, or inserted into, an expression vector.Recipient cells capable of expressing the chimeric immunoglobulin chaingene product are then transfected singly with an antibody,antigen-binding portion thereof, or CAR, or chimeric H or chimeric Lchain-encoding gene, or are co-transfected with a chimeric H and achimeric L chain gene. The transfected recipient cells are culturedunder conditions that permit expression of the incorporated genes andthe expressed immunoglobulin chains or intact antibodies or fragmentsare recovered from the culture.

In some embodiments, the genes encoding the antibody, antigen-bindingportion thereof, CAR, or chimeric H and L chains, or portions thereofare assembled in separate expression vectors that are then used toco-transfect a recipient cell. Each vector can contain two selectablegenes, a first selectable gene designed for selection in a bacterialsystem and a second selectable gene designed for selection in aeukaryotic system, wherein each vector has a different pair of genes.This strategy results in vectors which first direct the production, andpermit amplification, of the genes in a bacterial system. The genes soproduced and amplified in a bacterial host are subsequently used toco-transfect a eukaryotic cell, and allow selection of a co-transfectedcell carrying the desired transfected genes. Non-limiting examples ofselectable genes for use in a bacterial system are the gene that confersresistance to ampicillin and the gene that confers resistance tochloramphenicol. Selectable genes for use in eukaryotic transfectantsinclude the xanthine guanine phosphoribosyl transferase gene (designatedgpt) and the phosphotransferase gene from Tn5 (designated neo).Alternatively the genes can be assembled on the same expression vector.

For transfection of the expression vectors and production of theantibodies, antibody reagents, antigen-binding portions thereof, or CARSdescribed herein, the recipient cell line can be a myeloma cell. Myelomacells can synthesize, assemble and secrete immunoglobulins encoded bytransfected immunoglobulin genes and possess the mechanism forglycosylation of the immunoglobulin. For example, in some embodiments,the recipient cell is the recombinant Ig-producing myeloma cell SP2/0(ATCC #CRL 8287). SP2/0 cells produce only immunoglobulin encoded by thetransfected genes. Myeloma cells can be grown in culture or in theperitoneal cavity of a mouse, where secreted immunoglobulin can beobtained from ascites fluid. Other suitable recipient cells includelymphoid cells such as B lymphocytes of human or non-human origin,hybridoma cells of human or non-human origin, or interspeciesheterohybridoma cells.

An expression vector carrying a chimeric, humanized, or composite humanantibody construct, antibody, antigen-binding portion thereof, and/orCAR as described herein can be introduced into an appropriate host cellby any of a variety of suitable means, including such biochemical meansas transformation, transfection, conjugation, protoplast fusion, calciumphosphate-precipitation, and application with polycations such asdiethylaminoethyl (DEAE) dextran, and such mechanical means aselectroporation, direct microinjection, and microprojectile bombardment,as known to one of ordinary skill in the art.

Traditionally, monoclonal antibodies have been produced as nativemolecules in murine hybridoma lines. In addition to that technology, themethods and compositions described herein provide for recombinant DNAexpression of monoclonal antibodies. This allows the production ofhumanized antibodies as well as a spectrum of antibody derivatives andfusion proteins in a host species of choice. The production ofantibodies in bacteria, yeast, transgenic animals and chicken eggs arealso alternatives for hybridoma-based production systems. The mainadvantages of transgenic animals are potential high yields fromrenewable sources.

In one aspect, a cell comprising an isolated antibody, antigen-bindingportion thereof, or CAR as described herein is provided. In someembodiments, the isolated antibody, antigen-binding portion thereof, orCAR as described herein is expressed on the cell surface. In someembodiments, the cell comprises a nucleic acid encoding an isolatedantibody, antigen-binding portion thereof, or CAR as described herein.

In some embodiments, the cell is an immune cell. As used herein, “immunecell” refers to a cell that plays a role in the immune response. Immunecells are of hematopoietic origin, and include lymphocytes, such as Bcells and T cells; natural killer cells; myeloid cells, such asmonocytes, macrophages, eosinophils, mast cells, basophils, andgranulocytes. In some embodiments, the cell is a T cell; a NK cell; aNKT cell; lymphocytes, such as B cells and T cells; and myeloid cells,such as monocytes, macrophages, eosinophils, mast cells, basophils, andgranulocytes.

In particular embodiments, a cell (e.g., an immune cell) is transducedwith a retroviral vector, e.g., a lentiviral vector, encoding a CAR. Forexample, an immune effector cell is transduced with a vector encoding aCAR that comprises an anti-CHI3L1 antibody or antigen binding portionthereof that binds a CHI3L1 polypeptide, with an intracellular signalingdomain of CD3, CD28, 4-1BB, Ox40, or any combinations thereof. Thus,these transduced cells can elicit a CAR-mediated cytotoxic response.

Retroviruses are a common tool for gene delivery. In particularembodiments, a retrovirus is used to deliver a polynucleotide encoding achimeric antigen receptor (CAR) to a cell. As used herein, the term“retrovirus” refers to an RNA virus that reverse transcribes its genomicRNA into a linear double-stranded DNA copy and subsequently covalentlyintegrates its genomic DNA into a host genome. Once the virus isintegrated into the host genome, it is referred to as a “provirus.” Theprovirus serves as a template for RNA polymerase II and directs theexpression of RNA molecules which encode the structural proteins andenzymes needed to produce new viral particles.

Illustrative retroviruses suitable for use in particular embodiments,include, but are not limited to: Moloney murine leukemia virus (M-MuLV),Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus(GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemiavirus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) andlentivirus.

As used herein, the term “lentivirus” refers to a group (or genus) ofcomplex retroviruses. Illustrative lentiviruses include, but are notlimited to: HIV (human immunodeficiency virus; including HIV type 1, andHIV type 2); visna-maedi virus (VMV) virus; the caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (FIV); bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV). In one embodiment,HIV based vector backbones (i.e., HIV cis-acting sequence elements) arepreferred. In particular embodiments, a lentivirus is used to deliver apolynucleotide comprising a CAR to a cell.

Retroviral vectors and more particularly lentiviral vectors may be usedin practicing particular embodiments of the present invention.Accordingly, the term “retrovirus” or “retroviral vector”, as usedherein is meant to include “lentivirus” and “lentiviral vectors”respectively.

In one aspect of any of the embodiments, described herein is acompositionscomprising an antibody, antibody reagent, antigen-bindingportion thereof, or CAR as described herein or a nucleic acid encodingan antibody, antibody reagent, antigen-binding portion thereof, or CARas described herein or a cell as described herein. In some embodiments,the composition is a pharmaceutical composition. As used herein, theterm “pharmaceutical composition” refers to the active agent incombination with a pharmaceutically acceptable carrier accepted for usein the pharmaceutical industry. The phrase “pharmaceutically acceptable”is employed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance ormaintain the effectiveness of the active ingredient. The therapeuticcomposition as described herein can include pharmaceutically acceptablesalts of the components therein. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. Physiologically tolerablecarriers are well known in the art. Exemplary liquid carriers aresterile aqueous solutions that contain no materials in addition to theactive ingredients and water, or contain a buffer such as sodiumphosphate at physiological pH value, physiological saline or both, suchas phosphate-buffered saline. Still further, aqueous carriers cancontain more than one buffer salt, as well as salts such as sodium andpotassium chlorides, dextrose, polyethylene glycol and other solutes.Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions. The amount of an active agent used in the invention that willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques.

In some embodiments, the composition comprising an antibody, antibodyreagent, antigen-binding portion thereof, or CAR as described herein ora nucleic acid encoding an antibody, antibody reagent, antigen-bindingportion thereof, or CAR as described herein can be a lyophilisate.

In some embodiments, the technology described herein relates to asyringe or catheter, including an organ-specific catheter (e.g., renalcatheter, biliary catheter, cardiac catheter, etc.), comprising atherapeutically effective amount of a composition described herein.

As used herein, the phrase “therapeutically effective amount”,“effective amount” or “effective dose” refers to an amount that providesa therapeutic or aesthetic benefit in the treatment, prevention, ormanagement of a tumor or malignancy, e.g., an amount that provides astatistically significant decrease in at least one symptom, sign, ormarker of a tumor or malignancy. Determination of a therapeuticallyeffective amount is well within the capability of those skilled in theart. Generally, a therapeutically effective amount can vary with thesubject's history, age, condition, sex, as well as the severity and typeof the medical condition in the subject, and administration of otherpharmaceutically active agents

In one aspect, described herein is a method of inhibiting or killing aCHI3L1+ cell, the method comprising contacting the cell with an isolatedantibody, antibody reagent, antigen-binding portion thereof, or CAR asdescribed herein, a nucleic acid encoding such polypeptides, a cellcomprising such a polypeptide or nucleic acid, or a compositioncomprising such a polypeptide or nucleic acid. Inhibiting a CHI3L1+ cellcan comprise inhibiting the metabolic activity, metastasis, and/orproliferation of the cell. Assays for measuring metabolic activity,metastasis (e.g., migration assays) and proliferation are well known inthe art. Similarly, assays for measuring killing of CHI3L1+ cells, e.g.,cell viability assays are well known in the art.

As used herein, a “CHI3L1+” cell is a cell expressing an increased levelof CHI3L1+, e.g., as compared to a healthy cell of the same type or anaverage level of CHI3L1 found in healthy cells of the same type. In someembodiments, an increased level of CHI3L1 can be a level which is atleast 1.5× the level found in a reference, e.g., 1.5×, 2×, 3×, 4×, 5× orgreater than the reference level.

In one aspect, the technology described herein relates to a methodcomprising administering an antibody, antibody reagent, antigen-bindingportion thereof, or CAR as described herein or a nucleic acid encodingan antibody, antibody reagent, antigen-binding portion thereof, or CARas described herein to a subject. In some embodiments, the subject is inneed of treatment for a cancer and/or malignancy. In some embodiments,the subject is in need of treatment for: prostate cancer, colon cancer,rectal cancer, ovarian cancer, kidney cancer, breast cancer,glioblastoma, melanoma, malignant melanoma, and lung cancer. In someembodiments, the method is a method of treating a subject. In someembodiments, the method is a method of treating a cancer in a subject.

In one aspect, the technology described herein relates to a methodcomprising administering an antibody, antibody reagent, antigen-bindingportion thereof, or CAR as described herein or a nucleic acid encodingan antibody, antibody reagent, antigen-binding portion thereof, or CARas described herein to a subject. In some embodiments, the method is amethod of treating asthma. In some embodiments, the subject is in needof treatment for asthma.

Asthma refers to a chronic inflammatory disease of the respiratorysystem in which the airway occasionally constricts, becomes inflamed,and is lined with excessive amounts of mucus, often in response to oneor more triggers. Asthma can be defined simply as reversible airwayobstruction in an individual over a period of time. Asthma can beallergic/atopic or non-allergic. Asthma is characterized by the presenceof cells such as eosinophils, mast cells, basophils, and activated Tlymphocytes in the airway walls. With chronicity of the process,secondary changes occur, such as thickening of basement membranes andfibrosis. The disease is characterized by increased airwayhyperresponsiveness to a variety of stimuli, and airway inflammation andconstriction. This airway narrowing causes symptoms such as wheezing,shortness of breath, chest tightness, and coughing. The airwayconstriction responds to bronchodilators. Between episodes, mostpatients feel well but can have mild symptoms and they can remain shortof breath after exercise for longer periods of time than the unaffectedindividual. The symptoms of asthma can range from mild to lifethreatening.

Asthma can be triggered by such things as exposure to an allergen(allergic asthma), or non-allergens (non-allergic asthma) such as coldair, pollution (e.g., ozone), warm air, moist air, exercise or exertion,or emotional stress. In children, the most common triggers are viralillnesses such as those that cause the common cold (Zhao J., et. al.,2002, J Pediatr. Allergy Immunol. 13: 47-50).

Common allergens that trigger the allergic asthma include “seasonal”pollens, year-round dust mites, molds, pets, and insect parts, foods,such as fish, egg, peanuts, nuts, cow's milk, and soy, additives, suchas sulfites, work-related agents, such as latex. Approximately 80% ofchildren and 50% of adults with asthma also have allergies.

Common irritants that can trigger asthma in airways that arehyperreactive include respiratory infections, such as those caused byviral “colds,” bronchitis, and sinusitis, medication drugs, such asaspirin, other NSAIDs (nonsteroidal antiinflammatory drugs), and betablockers (used to treat blood pressure and other heart conditions),tobacco smoke, outdoor factors such as ozone, smog, weather changes, anddiesel fumes; indoor factors such as paint, detergents, deodorants,chemicals, and perfumes; nighttime GERD (gastroesophageal refluxdisorder); exercise, especially under cold dry conditions; work-relatedfactors such as chemicals, dusts, gases, and metals; emotional factors,such as laughing, crying, yelling, and distress; and hormonal factors,such as in premenstrual syndrome.

Regardless of the trigger, asthma is associated with reversible airwayobstruction and airway hyperreactivity (AHR), an increased sensitivityof the airways to nonspecific stimuli such as cold air or respiratoryirritants, and can be quantitated by responsiveness to methacholine orhistamine. A patient diagnosed as asthmatic will generally have multipleindications over time, including wheezing, asthmatic attacks, and apositive response to methacholine challenge, i.e., a PC20 onmethacholine challenge of less than about 4 mg/ml. The basic diagnosisand measurement of asthma is peak flow rates and the followingdiagnostic criteria are used by the British Thoracic Society (PinnockH., and Shah R., 2007, Br. Med. J. 334 (7598): 847-50): ≥20% differenceon at least three days in a week for at least two weeks; ≥20%improvement of peak flow following treatment, for example:10 minutes ofinhaled β-agonist (e.g., salbutamol), six week of inhaled corticosteroid(e.g., beclometasone), and 14 days of 30 mg prednisolone; and ≥20%decrease in peak flow following exposure to a trigger (e.g., exercise).Further guidelines for diagnosis may be found, for example, in theNational Asthma Education Program Expert Panel Guidelines for Diagnosisand Management of Asthma, National Institutes of Health, 1991, Pub. No.91-3042.

As demonstrated herein, the antibodies, antibody reagents, and/orantigen-binding portions thereof which are described herein can preventweight gain, e.g., when administered to subjects consuming high fatdiet. Accordingly, administration of the the antibodies, antibodyreagents, and/or antigen-binding portions thereof which are describedherein are contemplated for use in methods of treating obesity, reducingweight gain, preventing weight gain, promoting weight loss, and thelike. Such methods can, e.g., promote metabolic health, be pursued foraesthetic reasons, and/or prepare patients for surgical interventionswhich are counterindicated for those with high BMIs or weights.

In one aspect, the technology described herein relates to a methodcomprising administering an antibody, antibody reagent, antigen-bindingportion thereof, or CAR as described herein or a nucleic acid encodingan antibody, antibody reagent, antigen-binding portion thereof, or CARas described herein to a subject. In some embodiments, the method is amethod of treating obesity and/or preventing weight gain. In someembodiments, the subject is in need of treatment for obesity and/orweight gain. In some embodiments, the method is a method of treatingNASH, NFALD, a fatty liver disease, and/or metabolic syndrome. In someembodiments, the subject is in need of treatment for NASH, NFALD, afatty liver disease, and/or metabolic syndrome.

In one aspect, the technology described herein relates to a method ofpromoting weight and/or fat loss comprising administering an antibody,antibody reagent, antigen-binding portion thereof, or CAR as describedherein or a nucleic acid encoding an antibody, antibody reagent,antigen-binding portion thereof, or CAR as described herein to asubject. In some embodiments, the subject is a subject in need ofpromotion of weight loss.

The term “obesity” refers to excess fat in the body. Obesity can bedetermined by any measure accepted and utilized by those of skill in theart. Currently, an accepted measure of obesity is body mass index (BMI),which is a measure of body weight in kilograms relative to the square ofheight in meters. Generally, for an adult over age 20, a BMI betweenabout 18.5 and 24.9 is considered normal, a BMI between about 25.0 and29.9 is considered overweight, a BMI at or above about 30.0 isconsidered obese, and a BMI at or above about 40 is considered morbidlyobese. (See, e.g., Gallagher et al. (2000) Am J Clin Nutr 72:694-701.)These BMI ranges are based on the effect of body weight on increasedrisk for disease. Some common conditions related to high BMI and obesityinclude cardiovascular disease, high blood pressure (i.e.,hypertension), osteoarthritis, cancer, and diabetes. Although BMIcorrelates with body fat, the relation between BMI and actual body fatdiffers with age and gender. For example, women are more likely to havea higher percent of body fat than men for the same BMI. Furthermore, theBMI threshold that separates normal, overweight, and obese can vary,e.g. with age, gender, ethnicity, fitness, and body type, amongst otherfactors. In some embodiments, a subject with obesity can be a subjectwith a body mass index of at least about 25 kg/m² prior toadministration of a treatment as described herein. In some embodiments,a subject with obesity can be a subject with a body mass index of atleast about 30 kg/m² prior to administration of a treatment as describedherein.

The term “nonalcoholic fatty liver disease” or “NAFLD” describes a widespectrum of liver diseases ranging from simple fatty liver (steatosis)to nonalcoholic steatohepatitis (NASH) with progressive fibrosis andliver failure to cirrhosis. All of the stages of NAFLD have in commonthe accumulation of fat in the hepatocytes. In NASH, the fataccumulation is associated with varying degrees of inflammation(hepatitis) and scarring (fibrosis) of the liver. NAFLD and NASH canoccur in individuals who do not consume excessive amounts of alcohol.Yet, in many respects, the histological picture of an NAFLD biopsy issimilar to what can be seen in liver disease caused by alcohol abuse.NAFLD and NASH are considered the primary fatty liver diseases. Thesecondary fatty liver diseases include those that occur in other typesof liver disease. Secondary fatty liver can also occur in chronic viralhepatitis C (HCV), chronic viral hepatitis B (HBV), chronic autoimmunehepatitis (AIH), and Wilson's disease.

Hyperglycemia with or without evidence of hyperlipidemia is commonlyassociated with NAFLD. The disease exhibits the histological features ofalcohol-induced liver disease in patients who do not consume significantamounts of alcohol. All of the stages of NAFLD have in common theaccumulation of fat in the liver cells. Farrell and Larter inHepatology, 243:S99-S1 12 (2006) describe NASH as “the lynchpin” betweenhepatic steatosis and cirrhosis in the spectrum of NAFLD. See also,Palekar, et al., Liver mt., 26(2):151-6 (2006). In NASH, the fataccumulation of associated with varying degrees of inflammation andfibrosis. Conditions most commonly associated with NAFLD are obesity,type II diabetes and metabolic syndrome.

The symptoms of NAFLD and NASH can be identical. They are usually notdramatic and tend to be non-specific (as can also be observed in otherdiseases). The symptoms are minimal in most patients, who may, however,experience occasional, vague right upper-quadrant abdominal pain. Thispain characteristically is dull and aching, without a predictablepattern of occurrence. It is not an intense, sudden, and severe pain, asmight occur with, for example, gallstones. The abdominal pain in NAFLDand NASH is thought to be due to the stretching of the liver covering(capsule) when the liver enlarges and/or when there is inflammation inthe liver. In contrast to ALD, hepatitis B, or hepatitis C, symptoms ofsevere, acute liver failure (e.g. jaundice, intense fatigue, loss ofappetite, nausea, vomiting, and confusion) are not typically observed inNAFLD or NASH. Obesity and related conditions (e.g. diabetes,hypertension) are frequent seen among those suffering from NAFLD orNASH, and the classic signs of insulin resistance often dominate thephysical exam in NAFLD and NASH. Acanthosis nigricans, a darkpigmentation of the skin of the armpits and neck can be a sign ofinsulin resistance and is frequently seen in children with NASH. Whenthe liver is palpated, it usually feels normal. However, when very largeamounts of fat accumulate in the liver, it can be become quite largewith a soft, rounded edge that can be easily felt by the doctor.

In addition to the symptoms described above, a diagnosis of NAFLD orNASH is made based on the following criteria: clinical and/orbiochemical signs of insulin resistance; biopsy; chronically elevatedALT; signs of fatty liver on ultrasound; exclusion of other causes ofelevated ALT and fatty liver.

The term “steatosis” also referred to in the art as “fatty change” isthe process referring to the abnormal retention of lipids within a cell.It reflects an impairment of the normal processes of synthesis andbreakdown of triglyceride fat. Excess lipid accumulates in vesicles thatdisplace the cytoplasm. When the vesicles are large enough to distortthe nucleus, the condition is known as macrovesicular steatosis,otherwise the condition is known as microvesicular steatosis. Whilst notparticularly detrimental to the cell in mild cases, large accumulationscan disrupt cell constituents, and in severe cases the cell may evenburst.

The term “metabolic syndrome” refers to a combination of risk factorsfor cardiovascular disease (CVD) identified in the National CholesterolEducation Program's Adult Treatment Panel III report. See for examplethe discussion by Grundy et al in Circulation, 109 (2004), 433-438 whichis incorporated by reference herein in its entirety. The components ofmetabolic syndrome are: 1) abdominal obesity; 2) atherogenicdyslipidemia; 3) raised blood pressure; 4) insulin resistance; 5)proinflammatory state; and 6) prothrombotic state.

The term “fatty liver disease” as used herein refers to and comprisesall kinds of disorders as a consequence of fat accumulation and/or fatinfiltration into the liver that affect the anatomy, physiology,metabolism, and/or genetic activities of the liver, or that affect thegeneration of new liver cells and/or the regeneration of the liver, as awhole or parts thereof, transiently, temporarily, chronically orpermanently, in a pathological way.

The term “liver disease” refers to disorders that affect the anatomy ormetabolism of normal functioning of the liver. Liver diseases are causedby, for example, alcohol (e.g. ASH), non-alcoholic fatty liver changes(such as NAFLD including NASH), nutrition-mediated liver injury (forexample starvation), other toxic liver injury (such as unspecifichepatitis induced by e.g. drugs such as but not limited to acetaminophen(paracetamol), chlorinated hydrocarbons (e.g. CC14), amiodarone(cordarone), valproate, tetracycline (only i.v.), isoniacid (Druginduced liver disease 2004. Lazerow S K, Abdi M S, Lewis J H. Curr OpinGastroenterol., 2005, 21(3): 283-292), or food intoxication resulting inacute or chronic liver failure, e.g. by consumption of mushroomscontaining aflatoxins (preferably B1 aflatoxin) or ingestion of certainmetal (such as copper or cadmium) or herbal products used in naturalmedicine (hompeoatics such as Mild thistle, Chaparral, Kawa-Kawa),interference of bilirubin metabolism, hepatitis like syndromes,cholestasis, granulomatous lesions, intrahepatic vascular lesions andcirrhosis), trauma and surgery (e.g. Pringle maneuver),radiation-mediated liver injury (such as caused by radiotherapy).

In one aspect, described herein is a method of treating cancer in asubject in need thereof, the method comprising administering a cell asdescribed herein, e.g., a cell comprising an antibody, antibody reagent,antigen-binding portion thereof, or CAR as described herein. In someembodiments, the cell is an immune cell.

In one aspect, described herein is a method of treating cancer in asubject in need thereof, the method comprising administering a nucleicacid as described herein or an immune cell comprising the nucleic acidto the subject, wherein the subject's immune cells are caused to expressthe polypeptide encoded by the nucleic acid. In some embodiments, theimmune cell is a T cell. Nucleic acids can be targeted to particularcell types by, e.g., use of a cell-type specific promoter and/or acomposition that selectively binds to the desired cell type. Forexample, conjugation of a nucleic acid to an aptamer can permit targeteddelivery (McNamara, J O., et al. (2006) Nat. Biotechnol. 24:1005-1015).In an alternative embodiment, the nucleic acid can be delivered usingdrug delivery systems such as a nanoparticle, a dendrimer, a polymer,liposomes, or a cationic delivery system. Positively charged cationicdelivery systems facilitate binding of an nucleic acid molecule(negatively charged) and also enhance interactions at the negativelycharged cell membrane to permit efficient uptake of a nucleic acid bythe cell. Cationic lipids, dendrimers, or polymers can either be boundto a nucleic acid, or induced to form a vesicle or micelle (see e.g.,Kim S H., et al. (2008) Journal of Controlled Release 129(2):107-116)that encases a nucleic acid. The formation of vesicles or micellesfurther prevents degradation of the nucleic acid when administeredsystemically. Methods for making and administering cationic-inhibitorynucleic acid complexes are well within the abilities of one skilled inthe art. Some non-limiting examples of drug delivery systems useful forsystemic delivery of nucleic acids include DOTAP Oligofectamine, “solidnucleic acid lipid particles”, cardiolipin, polyethyleneimine,Arg-Gly-Asp (RGD) peptides, and polyamidoamines. In some embodiments, anucleic acid forms a complex with cyclodextrin for systemicadministration. Methods for administration and pharmaceuticalcompositions of nucleic acids and cyclodextrins can be found in U.S.Pat. No. 7,427,605, which is herein incorporated by reference in itsentirety. Targeted delivery of nucleic acids is described, for examplein Ikeda and Taira Pharmaceutical Res 2006 23:1631-1640; Soutschek etal., Nature 2004 432:173-8 and Lorenze et al. Bioorg. Med. Chem. Lett.14, 4975-4977 (2004); each of which is incorporated by reference hereinin its entirety. By way of example, the nucleic acid can be targeted toimmune cells by encapsulating the inhibitor in a liposome comprisingligands of receptors expressed on immune cells, e.g., TCRs. In someembodiments, the liposome can comprise aptamers specific for immunecells.

In some embodiments, the methods described herein relate to CAR-T celltherapy. CAR-T cell and related therapies relate to adoptive celltransfer of immune cells (e.g., T cells) expressing a CAR that bindsspecifically to a targeted cell type (e.g., cancer cells) to treat asubject. In some embodiments, the cells administered as part of thetherapy can be autologous to the subject. In some embodiments, the cellsadministered as part of the therapy are not autologous to the subject.In some embodiments, the cells are engineered and/or geneticallymodified to express the CAR. Further discussion of CAR-T therapies canbe found, e.g., in Maus et al. Blood 2014 123:2624-35; Reardon et al.Neuro-Oncology 2014 16:1441-1458; Hoyos et al. Haematologica 201297:1622; Byrd et al. J Clin Oncol 2014 32:3039-47; Maher et al. CancerRes 2009 69:4559-4562; and Tamada et al. Clin Cancer Res 201218:6436-6445; each of which is incorporated by reference herein in itsentirety.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues. Cancer cells can also spread to otherparts of the body through the blood and lymph systems. There are severalmain types of cancer. Carcinoma is a cancer that begins in the skin orin tissues that line or cover internal organs. Sarcoma is a cancer thatbegins in bone, cartilage, fat, muscle, blood vessels, or otherconnective or supportive tissue. Leukemia is a cancer that starts inblood-forming tissue such as the bone marrow, and causes large numbersof abnormal blood cells to be produced and enter the blood. Lymphoma andmultiple myeloma are cancers that begin in the cells of the immunesystem. Central nervous system cancers are cancers that begin in thetissues of the brain and spinal cord.

In some embodiments of any of the aspects, the cancer is a primarycancer. In some embodiments of any of the aspects, the cancer is amalignant cancer. As used herein, the term “malignant” refers to acancer in which a group of tumor cells display one or more ofuncontrolled growth (i.e., division beyond normal limits), invasion(i.e., intrusion on and destruction of adjacent tissues), and metastasis(i.e., spread to other locations in the body via lymph or blood). Asused herein, the term “metastasize” refers to the spread of cancer fromone part of the body to another. A tumor formed by cells that havespread is called a “metastatic tumor” or a “metastasis.” The metastatictumor contains cells that are like those in the original (primary)tumor.

As used herein, the term “benign” or “non-malignant” refers to tumorsthat may grow larger but do not spread to other parts of the body.Benign tumors are self-limited and typically do not invade ormetastasize.

A “cancer cell” or “tumor cell” refers to an individual cell of acancerous growth or tissue. A tumor refers generally to a swelling orlesion formed by an abnormal growth of cells, which may be benign,pre-malignant, or malignant. Most cancer cells form tumors, but some,e.g., leukemia, do not necessarily form tumors. For those cancer cellsthat form tumors, the terms cancer (cell) and tumor (cell) are usedinterchangeably.

A subject that has a cancer or a tumor is a subject having objectivelymeasurable cancer cells present in the subject's body. Included in thisdefinition are malignant, actively proliferative cancers, as well aspotentially dormant tumors or micrometastatses. Cancers which migratefrom their original location and seed other vital organs can eventuallylead to the death of the subject through the functional deterioration ofthe affected organs. Hemopoietic cancers, such as leukemia, are able toout-compete the normal hemopoietic compartments in a subject, therebyleading to hemopoietic failure (in the form of anemia, thrombocytopeniaand neutropenia) ultimately causing death.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancerof the peritoneum; cervical cancer; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelialneoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer;lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; as well as other carcinomas and sarcomas; as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

A “cancer cell” is a cancerous, pre-cancerous, or transformed cell,either in vivo, ex vivo, or in tissue culture, that has spontaneous orinduced phenotypic changes that do not necessarily involve the uptake ofnew genetic material. Although transformation can arise from infectionwith a transforming virus and incorporation of new genomic nucleic acid,or uptake of exogenous nucleic acid, it can also arise spontaneously orfollowing exposure to a carcinogen, thereby mutating an endogenous gene.Transformation/cancer is associated with, e.g., morphological changes,immortalization of cells, aberrant growth control, foci formation,anchorage independence, malignancy, loss of contact inhibition anddensity limitation of growth, growth factor or serum independence, tumorspecific markers, invasiveness or metastasis, and tumor growth insuitable animal hosts such as nude mice.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patients or subjects include any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments, the subject is a mammal,e.g., a primate, e.g., a human. The terms, “patient”, “individual” and“subject” are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used, for example, as subjects that represent animalmodels of, for example, various cancers. In addition, the methodsdescribed herein can be used to treat domesticated animals and/or pets.A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g., a cancer) or one or more complications related to such acondition, and optionally, but need not have already undergone treatmentfor a condition or the one or more complications related to thecondition. Alternatively, a subject can also be one who has not beenpreviously diagnosed as having a condition in need of treatment or oneor more complications related to such a condition. For example, asubject can be one who exhibits one or more risk factors for a conditionor one or more complications related to a condition or a subject whodoes not exhibit risk factors. A “subject in need” of treatment for aparticular condition can be a subject having that condition, diagnosedas having that condition, or at risk of developing that condition.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” when used in reference to a disease, disorder or medicalcondition, refer to therapeutic treatments for a condition, wherein theobject is to reverse, alleviate, ameliorate, inhibit, slow down or stopthe progression or severity of a symptom or condition. The term“treating” includes reducing or alleviating at least one adverse effector symptom of a condition. Treatment is generally “effective” if one ormore symptoms or clinical markers are reduced. Alternatively, treatmentis “effective” if the progression of a condition is reduced or halted.That is, “treatment” includes not just the improvement of symptoms ormarkers, but also a cessation or at least slowing of progress orworsening of symptoms that would be expected in the absence oftreatment. Beneficial or desired clinical results include, but are notlimited to, alleviation of one or more symptom(s), diminishment ofextent of the deficit, stabilized (i.e., not worsening) state of a tumoror malignancy, delay or slowing of tumor growth and/or metastasis, andan increased lifespan as compared to that expected in the absence oftreatment. As used herein, the term “administering,” refers to theplacement of an agent, including but not limited to, an antibody,antibody reagent, antigen-binding portion thereof, or CAR as describedherein or a nucleic acid encoding an antibody, antibody reagent,antigen-binding portion thereof, or CAR, or a cell comprising such anagent, as described herein into a subject by a method or route whichresults in at least partial localization of the agents at a desiredsite. The pharmaceutical composition comprising an antibody, antibodyreagent, antigen-binding portion thereof, or CAR as described herein ora nucleic acid encoding an antibody, antibody reagent, antigen-bindingportion thereof, or CAR, or a cell comprising such an agent as describedherein disclosed herein can be administered by any appropriate routewhich results in an effective treatment in the subject.

The administration of the compositions contemplated herein may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. In apreferred embodiment, compositions are administered parenterally. Thephrases “parenteral administration” and “administered parenterally” asused herein refers to modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravascular, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intratumoral, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion. In one embodiment, the compositions contemplatedherein are administered to a subject by direct injection into a tumor,lymph node, or site of infection.

It can generally be stated that a pharmaceutical composition comprisingthe cells, e.g., T cells or immune cells, described herein may beadministered at a dosage of 10² to 10¹⁰ cells/kg body weight, preferably10⁵ to 10⁶ cells/kg body weight, including all integer values withinthose ranges. The number of cells will depend upon the ultimate use forwhich the composition is intended as will the type of cells includedtherein. For uses provided herein, the cells are generally in a volumeof a liter or less, can be 500 mLs or less, even 250 mLs or 100 mLs orless. Hence the density of the desired cells is typically greater than10⁶ cells/ml and generally is greater than 10⁷ cells/ml, generally 10⁸cells/ml or greater. The clinically relevant number of immune cells canbe apportioned into multiple infusions that cumulatively equal or exceed10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² cells. In some aspects ofthe present invention, particularly since all the infused cells will beredirected to a particular target antigen, lower numbers of cells, inthe range of 10⁶/kilogram (10⁶-10¹¹ per patient) may be administered.CAR expressing cell compositions may be administered multiple times atdosages within these ranges. The cells may be allogeneic, syngeneic,xenogeneic, or autologous to the patient undergoing therapy. If desired,the treatment may also include administration of mitogens (e.g., PHA) orlymphokines, cytokines, and/or chemokines (e.g., IFN-γ, IL-2, IL-12,TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES,MIP1α, etc.) as described herein to enhance induction of the immuneresponse. In some embodiments, the dosage can be from about 1×10⁵ cellsto about 1×10⁸ cells per kg of body weight. In some embodiments, thedosage can be from about 1×10⁶ cells to about 1×10⁷ cells per kg of bodyweight. In some embodiments, the dosage can be about 1×10⁶ cells per kgof body weight. In some embodiments, one dose of cells can beadministered. In some embodiments, the dose of cells can be repeated,e.g., once, twice, or more. In some embodiments, the dose of cells canbe administered on, e.g., a daily, weekly, or monthly basis.

The dosage ranges for the agent depend upon the potency, and encompassamounts large enough to produce the desired effect e.g., slowing oftumor growth or a reduction in tumor size. The dosage should not be solarge as to cause unacceptable adverse side effects. Generally, thedosage will vary with the age, condition, and sex of the patient and canbe determined by one of skill in the art. The dosage can also beadjusted by the individual physician in the event of any complication.In some embodiments, the dosage ranges from 0.001 mg/kg body weight to0.5 mg/kg body weight. In some embodiments, the dose range is from 5μg/kg body weight to 100 μg/kg body weight. Alternatively, the doserange can be titrated to maintain serum levels between 1 μg/mL and 1000μg/mL. For systemic administration, subjects can be administered atherapeutic amount, such as, e.g., 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30mg/kg, 40 mg/kg, 50 mg/kg, or more.

Administration of the doses recited above can be repeated. In someembodiments, the doses are given once a day, or multiple times a day,for example but not limited to three times a day. In a some embodiments,the doses recited above are administered daily for several weeks ormonths. The duration of treatment depends upon the subject's clinicalprogress and responsiveness to therapy.

In some embodiments, the dose can be from about 2 mg/kg to about 15mg/kg. In some embodiments, the dose can be about 2 mg/kg. In someembodiments, the dose can be about 4 mg/kg. In some embodiments, thedose can be about 5 mg/kg. In some embodiments, the dose can be about 6mg/kg. In some embodiments, the dose can be about 8 mg/kg. In someembodiments, the dose can be about 10 mg/kg. In some embodiments, thedose can be about 15 mg/kg. In some embodiments, the dose can be fromabout 100 mg/m² to about 700 mg/m². In some embodiments, the dose can beabout 250 mg/m². In some embodiments, the dose can be about 375 mg/m².In some embodiments, the dose can be about 400 mg/m². In someembodiments, the dose can be about 500 mg/m².

In some embodiments, the dose can be administered intravenously. In someembodiments, the intravenous administration can be an infusion occurringover a period of from about 10 minute to about 3 hours. In someembodiments, the intravenous administration can be an infusion occurringover a period of from about 30 minutes to about 90 minutes.

In some embodiments the dose can be administered about weekly. In someembodiments, the dose can be administered weekly. In some embodiments,the dose can be administered weekly for from about 12 weeks to about 18weeks. In some embodiments the dose can be administered about every 2weeks. In some embodiments the dose can be administered about every 3weeks. In some embodiments, the dose can be from about 2 mg/kg to about15 mg/kg administered about every 2 weeks. In some embodiments, the dosecan be from about 2 mg/kg to about 15 mg/kg administered about every 3weeks. In some embodiments, the dose can be from about 2 mg/kg to about15 mg/kg administered intravenously about every 2 weeks. In someembodiments, the dose can be from about 2 mg/kg to about 15 mg/kgadministered intravenously about every 3 weeks. In some embodiments, thedose can be from about 200 mg/m² to about 400 mg/m² administeredintravenously about every week. In some embodiments, the dose can befrom about 200 mg/m² to about 400 mg/m² administered intravenously aboutevery 2 weeks. In some embodiments, the dose can be from about 200 mg/m²to about 400 mg/m² administered intravenously about every 3 weeks. Insome embodiments, a total of from about 2 to about 10 doses areadministered. In some embodiments, a total of 4 doses are administered.In some embodiments, a total of 5 doses are administered. In someembodiments, a total of 6 doses are administered. In some embodiments, atotal of 7 doses are administered. In some embodiments, a total of 8doses are administered. In some embodiments, the administration occursfor a total of from about 4 weeks to about 12 weeks. In someembodiments, the administration occurs for a total of about 6 weeks. Insome embodiments, the administration occurs for a total of about 8weeks. In some embodiments, the administration occurs for a total ofabout 12 weeks. In some embodiments, the initial dose can be from about1.5 to about 2.5 fold greater than subsequent doses.

In some embodiments, the dose can be from about 1 mg to about 2000 mg.In some embodiments, the dose can be about 3 mg. In some embodiments,the dose can be about 10 mg. In some embodiments, the dose can be about30 mg. In some embodiments, the dose can be about 1000 mg. In someembodiments, the dose can be about 2000 mg. In some embodiments, thedose can be about 3 mg given by intravenous infusion daily. In someembodiments, the dose can be about 10 mg given by intravenous infusiondaily. In some embodiments, the dose can be about 30 mg given byintravenous infusion three times per week.

A therapeutically effective amount is an amount of an agent that issufficient to produce a statistically significant, measurable change intumor size, tumor growth etc. (efficacy measurements are described belowherein). Such effective amounts can be gauged in clinical trials as wellas animal studies.

An agent can be administered intravenously by injection or by gradualinfusion over time. Given an appropriate formulation for a given route,for example, agents useful in the methods and compositions describedherein can be administered intravenously, intranasally, by inhalation,intraperitoneally, intramuscularly, subcutaneously, intracavity, and canbe delivered by peristaltic means, if desired, or by other means knownby those skilled in the art. It is preferred that the compounds usedherein are administered orally, intravenously or intramuscularly to apatient having cancer. Local administration directly to a tumor mass isalso specifically contemplated.

Therapeutic compositions containing at least one agent can beconventionally administered in a unit dose, for example. The term “unitdose” when used in reference to a therapeutic composition refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required physiologically acceptable diluent, i.e., carrier, orvehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired.

Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are particular to eachindividual. However, suitable dosage ranges for systemic application aredisclosed herein and depend on the route of administration. Suitableregimes for administration are also variable, but are typified by aninitial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the blood in the ranges specified for in vivotherapies are contemplated.

In some embodiments, the methods further comprise administering thepharmaceutical composition described herein along with one or moreadditional chemotherapeutic agents, biologics, drugs, or treatments aspart of a combinatorial therapy. In some such embodiments, thechemotherapeutic agent biologic, drug, or treatment is selected from thegroup consisting of: radiation therapy, surgery, antibody reagents,and/or small molecules.

In some embodiments of the methods described herein, the methods furthercomprise administering one or more chemotherapeutic agents to thesubject being administered the pharmaceutical composition describedherein. Non-limiting examples of chemotherapeutic agents can includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1; dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any one of the above.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², ³²P and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins, such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

As used herein, the terms “chemotherapy” or “chemotherapeutic agent”refer to any chemical agent with therapeutic usefulness in the treatmentof diseases characterized by abnormal cell growth. Such diseases includetumors, neoplasms and cancer as well as diseases characterized byhyperplastic growth. Chemotherapeutic agents as used herein encompassboth chemical and biological agents. These agents function to inhibit acellular activity upon which the cancer cell depends for continuedsurvival. Categories of chemotherapeutic agents includealkylating/alkaloid agents, antimetabolites, hormones or hormoneanalogs, and miscellaneous antineoplastic drugs. Most if not all ofthese agents are directly toxic to cancer cells and do not requireimmune stimulation. In one embodiment, a chemotherapeutic agent is anagent of use in treating neoplasms such as solid tumors. In oneembodiment, a chemotherapeutic agent is a radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g., see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) Edition, 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds)). The bispecificand multispecific polypeptide agents described herein can be used inconjunction with additional chemotherapeutic agents.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone time administration and typical dosages range from 10 to 200 units(Grays) per day.

In some embodiments, the methods described herein can further compriseadministering an additional immunotherapy to the subject. As usedherein, “immunotherapy” refers to a diverse set of therapeuticstrategies designed to induce the patient's own immune system to fightthe tumor, and include, but are not limited to, intravesical BCGimmunotherapy for superficial bladder cancer, vaccines to generatespecific immune responses, such as for malignant melanoma and renal cellcarcinoma, the use of Sipuleucel-T for prostate cancer, in whichdendritic cells from the patient are loaded with prostatic acidphosphatase peptides to induce a specific immune response againstprostate-derived cells, administration of cytokines, growth factorsand/or signaling molecules that stimulate one or more immune cell type(e.g., interleukins), ex vivo expansion and/or stimulation oflymphocytes and/or dendritic cell specific for a tumor antigen prior toreintroduction to the patient, imiquimod, adoptive cell transfer, and/orthe methods described, e.g., in International Patent Publication WO2003/063792 and U.S. Pat. No. 8,329,660. In some embodiments, theimmunotherapy stimulates NK responses. In some embodiments, theimmunotherapy is an adoptive cell transfer approach, i.e., adoptiveimmunotherapy.

In some embodiments, the methods described herein can further compriseadministering an additional antibody, antibody reagent, antigen-bindingportion thereof, or T cell comprising a CAR to the subject. In someembodiments, the methods described herein can further compriseadministering cytokine to the subject. Antibody- and cytokine-basedtherapies are known in the art and can include, by way of non-limitingexample, alemtuzumab; bevacizumab; brentuximab vedotin; cetuximab;gemtuzumab; ibritumomab tiuxetan; ipilimumab; ofatumumab; pantibumumab;rituximab; tositumomab; trastuzumab; interleukin-2, andinterferon-alpha.

The efficacy of a given treatment for, e.g., cancer, can be determinedby the skilled clinician. However, a treatment is considered “effectivetreatment,” as the term is used herein, if any one or all of the signsor symptoms of e.g., a tumor are altered in a beneficial manner or otherclinically accepted symptoms are improved, or even ameliorated, e.g., byat least 10% following treatment with an agent as described herein.Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization or need for medical interventions (i.e.,progression of the disease is halted). Methods of measuring theseindicators are known to those of skill in the art and/or describedherein.

An effective amount for the treatment of a disease means that amountwhich, when administered to a mammal in need thereof, is sufficient toresult in effective treatment as that term is defined herein, for thatdisease. Efficacy of an agent can be determined by assessing physicalindicators of, for example cancer, e.g., tumor size, tumor mass, tumordensity, angiogenesis, tumor growth rate, etc.

In one aspect, described herein is a method of detecting, prognosing,and/or diagnosing cancer or asthma, the method comprising detecting ormeasuring the level of CHI3L1 in a sample obtained from a subject bycontacting the sample with an antibody, antibody reagent orantigen-binding portion thereof as described herein, wherein an increasein CHI3L1 levels relative to a reference level indicates the subject hascancer, is at increased risk of developing cancer or asthma.

In some embodiments of any of the aspects described herein, a subjectadministered a composition described herein can be a subject determinedto have an elevated level of CHI3L1. In some embodiments, the elevatedlevel of CHI3L1 is the level of circulating CHI3L1. In some embodimentsof any of the aspects described herein, a subject administered acomposition described herein can be a subject determined to have cancercells which are CHI3L1+.

In some embodiments of any of the aspects described herein, the methodcomprising administering a composition as described herein can furthercomprise a first step of identifying a subject having an elevated levelof CHI3L1. In some embodiments, the elevated level of CHI3L1 is thelevel of circulating CHI3L1. In some embodiments of any of the aspectsdescribed herein, the method comprising administering a composition asdescribed herein can further comprise a first step of identifying asubject having cancer cells which are CHI3L1+.

In one aspect, described herein is an assay comprising contacting a testsample obtained from the subject with an antibody, antibody reagent, orantigen-binding portion thereof as described herein, and detecting thepresence or intensity of a signal which indicates the presence or levelof CHI3L1 in the sample; wherein an increase in the CHI3L1 levelrelative to a reference level indicates the subject has a higher risk ofhaving or developing cancer.

In one aspect, described herein is a method of identifying a subject inneed of treatment for cancer, the method comprising: contacting a testsample obtained from the subject with an antibody, antibody reagent, orantigen-binding portion thereof as described herein, detecting thepresence or intensity of a signal which indicates the presence or levelof CHI3L1 in the sample; and identifying the subject as being in need oftreatment for cancer when the expression level CHI3L1 is increasedrelative to a reference level.

In one aspect, described herein is a method of determining if a subjectis likely to respond to treatment with anti-CHI3L1 therapy, e.g., ananti-CHI3L1 antibody, antibody reagent, or antigen binding portionthereof, or T cell comprising a CAR that binds CHI3L1, the methodcomprising: contacting a test sample obtained from the subject with anantibody, antibody reagent, or antigen-binding portion thereof asdescribed herein, detecting the presence or intensity of a signal whichindicates the presence or level of CHI3L1 in the sample; determiningthat the subject is likely to respond to treatment with anti-CHI3L1therapy when the level of CHI3L1 is increased relative to a referencelevel; and determining that the subject is not likely to respond totreatment with anti-CHI3L1 when the level of CHI3L1 is not increasedrelative to a reference level.

In one aspect, described herein is a method of treatment for cancercomprising; contacting a test sample obtained from the subject with anantibody, antibody reagent, or antigen-binding portion thereof asdescribed herein; detecting the presence or intensity of a signal whichindicates the presence or level of CHI3L1 in the sample; and treatingthe subject with an anti-CHI3L1 therapy when the level of CHI3L1 isincreased relative to a reference level. In one aspect, described hereinis a method of treating cancer comprising; administering atherapeutically effective amount of an anti-CHI3L1 therapy to a subjectdetermined to be in need of treatment for cancer and further determinedto have a level of CHI3L1 that is increased relative to a referencelevel, wherein the anti-CHI3L1 therapy comprises an antibody, antibodyreagent, antigen-binding portion thereof, or T cell comprising a CARthat recognizes CHI3L1; nucleic acid; cell; or composition as describedherein.

In one aspect, described herein is a method of detecting CHI3L1, themethod comprising contacting a biological sample with an antibody,antibody reagent, or antigen-binding portion thereof as describedherein, wherein reaction of the antibody or antigen-binding portionthereof with CHI3L1 indicates the presence of CHI3L1.

In some embodiments, the expression level of CHI3L1 can be measured bydetermining the level of an expression product of the CHI3L1 gene, e.g.,a CHI3L1 RNA transcript or a CHI3L1 polypeptide. Such molecules can beisolated, derived, or amplified from a biological sample, such as abiofluid. In some embodiments, a detectable signal is generated by theantibody or antigen-binding portion thereof when a CHI3L1 molecule ispresent. In some embodiments, the antibody or antigen-binding portionthereof is detectably labeled or capable of generating a detectablesignal. In some embodiments, the level of the CHI3L1 is determined usinga method selected from the group consisting of: Western blot;immunoprecipitation; enzyme-linked immunosorbent assay (ELISA);radioimmunological assay (RIA); sandwich assay; fluorescence in situhybridization (FISH); immunohistological staining; radioimmunometricassay; immunofluoresence assay; mass spectroscopy; FACS; andimmunoelectrophoresis assay. In some embodiments, the antibody orantigen-binding portion thereof is detectably labeled or generates adetectable signal. In some embodiments, the expression level of CHI3L1is normalized relative to the expression level of one or more referencegenes or reference proteins. In some embodiments, the reference level ofCHI3L1 is the expression level of CHI3L1 in a prior sample obtained fromthe subject.

In some embodiments, the level of CHI3L1 can be the level of CHI3L1polypeptide. Detection of CHI3L1 polypeptides can be according to anymethod known in the art. Immunological methods to detect CHI3L1polypeptides in accordance with the present technology include, but arenot limited to antibody techniques such as immunohistochemistry,immunocytochemistry, flow cytometry, fluorescence-activated cell sorting(FACS), immunoblotting, radioimmunoassays, western blotting,immunoprecipitation, enzyme-linked immunosorbant assays (ELISA), andderivative techniques that make use of antibody reagents as describedherein.

Immunochemical methods require the use of an antibody reagent specificfor the target molecule (e.g., the antigen or in the embodimentsdescribed herein, a CHI3L1 polypeptide. In some embodiments, the assays,methods, and/or systems described herein can comprise: an anti-CHI3L1antibody reagent. In some embodiments, the antibody reagent can bedetectably labeled. In some embodiments, the antibody reagent can beattached to a solid support (e.g., bound to a solid support). In someembodiments, the solid support can comprise a particle (including, butnot limited to an agarose or latex bead or particle or a magneticparticle), a bead, a nanoparticle, a polymer, a substrate, a slide, acoverslip, a plate, a dish, a well, a membrane, and/or a grating. Thesolid support can include many different materials including, but notlimited to, polymers, plastics, resins, polysaccharides, silicon orsilica based materials, carbon, metals, inorganic glasses, andmembranes.

In one embodiment, an assay, method, and/or system as described hereincan comprise an ELISA. In an exemplary embodiment, a first antibodyreagent can be immobilized on a solid support (usually a polystyrenemicro titer plate). The solid support can be contacted with a sampleobtained from a subject, and the antibody reagent will bind (“capture”)antigens for which it is specific (e.g., CHI3L1). The solid support canthen be contacted with a second labeled antibody reagent (e.g., adetection antibody reagent). The detection antibody reagent can, e.g.,comprise a detectable signal, be covalently linked to an enzyme, or canitself be detected by a secondary antibody which is linked to an enzymethrough bio-conjugation. The presence of a signal indicates that boththe first antibody reagent immobilized on the support and the second“detection” antibody reagent have bound to an antigen, i.e., thepresence of a signal indicated the presence of a CHI3L1 molecule.Between each step the plate is typically washed with a mild detergentsolution to remove any proteins or antibodies that are not specificallybound. After the final wash step the plate is developed by adding anenzymatic substrate to produce a visible signal, which indicates thequantity of CHI3L1 polypeptides in the sample. Older ELISAs utilizechromogenic substrates, though newer assays employ fluorogenicsubstrates with much higher sensitivity. There are other different formsof ELISA, which are well known to those skilled in the art.

In one embodiment, the assays, systems, and methods described herein cancomprise a lateral flow immunoassay test (LFIA), also known as theimmunochromatographic assay, or strip test to measure or determine thelevel of CHI3L1 polypeptide in a sample. LFIAs are a simple deviceintended to detect the presence (or absence) of CHI3L1 in a sample.There are currently many LFIA tests used for medical diagnostics eitherfor home testing, point of care testing, or laboratory use. LFIA testsare a form of immunoassay in which the test sample flows along a solidsubstrate via capillary action. After the sample is applied to the teststrip it encounters a colored antibody reagent which mixes with thesample, and if bound to a portion of the sample, transits the substrateencountering lines or zones which have been pretreated with a secondantibody reagent. Depending upon the level of CHI3L1 present in thesample the colored antibody reagent can become bound at the test line orzone. LFIAs are essentially immunoassays adapted to operate along asingle axis to suit the test strip format or a dipstick format. Striptests are extremely versatile and can be easily modified by one skilledin the art for detecting an enormous range of antigens from fluidsamples such as urine, blood, water samples etc. Strip tests are alsoknown as dip stick test, the name bearing from the literal action of“dipping” the test strip into a fluid sample to be tested. LFIA striptest are easy to use, require minimum training and can easily beincluded as components of point-of-care test (POCT) diagnostics to beused on site in the field. LFIA tests can be operated as eithercompetitive or sandwich assays. Sandwich LFIAs are similar to sandwichELISA. The sample first encounters colored particles which are labeledwith antibody reagents specific for a target (e.g., a CHI3L1-specificantibody reagent). The test line will also contain antibody reagents(e.g., a CHI3L1-specific antibody reagent). The test line will show as acolored band in positive samples. In some embodiments, the lateral flowimmunoassay can be a double antibody sandwich assay, a competitiveassay, a quantitative assay or variations thereof. There are a number ofvariations on lateral flow technology. It is also possible to applymultiple capture zones to create a multiplex test.

A typical test strip consists of the following components: (1) sampleapplication area comprising an absorbent pad (i. e. the matrix ormaterial) onto which the test sample is applied; (2) conjugate orreagent pad—this contains antibody reagent(s) specific to the targetwhich can be conjugated to colored particles (usually colloidal goldparticles, or latex microspheres); (3) test results area comprising areaction membrane—typically a hydrophobic nitrocellulose or celluloseacetate membrane onto which antibody reagents are immobilized in a lineacross the membrane as a capture zone or test line (a control zone mayalso be present, containing antibodies specific for the antibodyreagents conjugated to the particles or microspheres); and (4) optionalwick or waste reservoir—a further absorbent pad designed to draw thesample across the reaction membrane by capillary action and collect it.The components of the strip are usually fixed to an inert backingmaterial and may be presented in a simple dipstick format or within aplastic casing with a sample port and reaction window showing thecapture and control zones. While not strictly necessary, most tests willincorporate a second line which contains an antibody that picks up freelatex/gold in order to confirm the test has operated correctly.

The use of “dip sticks” or LFIA test strips and other solid supports hasbeen described in the art in the context of an immunoassay for a numberof antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598;5,770,460; 5,622,871; 6,565,808, U.S. patent application Ser. No.10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, whichare incorporated herein by reference in their entirety, are non-limitingexamples of such lateral flow test devices. Three U.S. patents (U.S.Pat. No. 4,444,880, issued to H. Tom; U.S. Pat. No. 4,305,924, issued toR. N. Piasio; and U.S. Pat. No. 4,135,884, issued to J. T. Shen)describe the use of “dip stick” technology to detect soluble antigensvia immunochemical assays. The apparatuses and methods of these threepatents broadly describe a first component fixed to a solid surface on a“dip stick” which is exposed to a solution containing a soluble antigenthat binds to the component fixed upon the “dip stick,” prior todetection of the component-antigen complex upon the stick. It is withinthe skill of one in the art to modify the teaching of these “dip stick”technologies as necessary for the detection of CHI3L1 polypeptides. Insome embodiments, the dip stick (or LFIA) can be suitable for use withurine samples. In some embodiments, a dip stick can be suitable for usewith blood samples.

Immunochemistry is a family of techniques based on the use of a specificantibody, wherein antibodies are used to specifically target moleculesinside or on the surface of cells. In some embodiments,immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniquescan be used to detect or measure the levels of CHI3L1 polypeptide. IHCis the application of immunochemistry to tissue sections, whereas ICC isthe application of immunochemistry to cells or tissue imprints afterthey have undergone specific cytological preparations such as, forexample, liquid-based preparations. In some instances, signalamplification may be integrated into the particular protocol, wherein asecondary antibody, that includes a label, follows the application of anantibody reagent specific for platelets or leukocytes. Typically, forimmunohistochemistry, tissue obtained from a subject and fixed by asuitable fixing agent such as alcohol, acetone, and paraformaldehyde, issectioned and reacted with an antibody. Conventional methods forimmunohistochemistry are described in Buchwalow and Bocker (Eds)“Immunohistochemistry: Basics and Methods” Springer (2010): Lin andPrichard “Handbook of Practical Immunohistochemistry” Springer (2011);which are incorporated by reference herein in their entireties. In someembodiments, immunocytochemistry may be utilized where, in general,tissue or cells obtained from a subject are fixed by a suitable fixingagent such as alcohol, acetone, and paraformaldehyde, to which isreacted an antibody. Methods of immunocytological staining of humansamples is known to those of skill in the art and described, forexample, in Burry “Immunocytochemistry: A Practical Guide for BiomedicalResearch” Springer (2009); which is incorporated by reference herein inits entirety.

In some embodiments, one or more of the antibody reagents describedherein can comprise a detectable label and/or comprise the ability togenerate a detectable signal (e.g., by catalyzing a reaction convertinga compound to a detectable product). Detectable labels can comprise, forexample, a light-absorbing dye, a fluorescent dye, or a radioactivelabel. Detectable labels, methods of detecting them, and methods ofincorporating them into an antibody reagent are well known in the art.

In some embodiments, detectable labels can include labels that can bedetected by spectroscopic, photochemical, biochemical, immunochemical,electromagnetic, radiochemical, or chemical means, such as fluorescence,chemifluoresence, or chemiluminescence, or any other appropriate means.The detectable labels used in the methods described herein can beprimary labels (where the label comprises a moiety that is directlydetectable or that produces a directly detectable moiety) or secondarylabels (where the detectable label binds to another moiety to produce adetectable signal, e.g., as is common in immunological labeling usingsecondary and tertiary antibodies). The detectable label can be linkedby covalent or non-covalent means to the antibody reagent.Alternatively, a detectable label can be linked such as by directlylabeling a molecule that achieves binding to the antibody reagent via aligand-receptor binding pair arrangement or other such specificrecognition molecules. Detectable labels can include, but are notlimited to radioisotopes, bioluminescent compounds, chromophores,antibodies, chemiluminescent compounds, fluorescent compounds, metalchelates, and enzymes.

In other embodiments, the detection antibody is labeled with afluorescent compound. When the fluorescently labeled antibody is exposedto light of the proper wavelength, its presence can then be detected dueto fluorescence. In some embodiments, a detectable label can be afluorescent dye molecule, or fluorophore including, but not limited tofluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine,Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine,tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein,rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™,rhodamine and derivatives (e.g., Texas red and tetrarhodimineisothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™,6-carboxyfhiorescein (commonly known by the abbreviations FAM and F),6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J),N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g., Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g., Hoechst 33258; phenanthridine dyes, e.g., Texas Red;ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes;porphyrin dyes; polymethine dyes, e.g., cyanine dyes such as Cy3, Cy5,etc; BODIPY dyes and quinoline dyes.

In some embodiments, a detectable label can be a radiolabel including,but not limited to ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and ³³P.

In some embodiments, a detectable label can be an enzyme including, butnot limited to horseradish peroxidase and alkaline phosphatase. Anenzymatic label can produce, for example, a chemiluminescent signal, acolor signal, or a fluorescent signal. Enzymes contemplated for use todetectably label an antibody reagent include, but are not limited to,malate dehydrogenase, staphylococcal nuclease, delta-V-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholinesterase.

In some embodiments, a detectable label is a chemiluminescent label,including, but not limited to lucigenin, luminol, luciferin, isoluminol,theromatic acridinium ester, imidazole, acridinium salt and oxalateester.

In some embodiments, a detectable label can be a spectral colorimetriclabel including, but not limited to colloidal gold or colored glass orplastic (e.g., polystyrene, polypropylene, and latex) beads.

In some embodiments, antibodies can also be labeled with a detectabletag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Otherdetection systems can also be used, for example, a biotin-streptavidinsystem. In this system, the antibodies immunoreactive (i. e. specificfor) with the biomarker of interest is biotinylated. Quantity ofbiotinylated antibody bound to the biomarker is determined using astreptavidin-peroxidase conjugate and a chromagenic substrate. Suchstreptavidin peroxidase detection kits are commercially available, e. g.from DAKO; Carpinteria, Calif.

An antibody reagent can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody reagent using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

The assays and methods as described herein can relate to determining ifa subject has an increased level of CHI3L1 relative to a referencelevel. In some embodiments, the reference level of CHI3L1 can be thelevel of CHI3L1 in a healthy subject not having, or not diagnosed ashaving, e.g., cancer. In some embodiments, the reference level can bethe level in a sample of similar cell type, sample type, sampleprocessing, and/or obtained from a subject of similar age, sex and otherdemographic parameters as the sample/subject for which the level ofCHI3L1 is to be determined. In some embodiments, the test sample andcontrol reference sample are of the same type, that is, obtained fromthe same biological source, and comprising the same composition, e.g.,the same number and type of cells and/or type of sample material.Accordingly, in some embodiments, the level of CHI3L1 which is increasedcan vary as demographic factors such as age, gender, genotype,environmental factors, and individual medical histories vary. In someembodiments, the reference level can comprise the level of CHI3L1 (e.g.,CHI3L1 polypeptide) in a sample of the same type taken from a subjectnot exhibiting any signs or symptoms of, e.g., cancer. In someembodiments, the reference expression level of CHI3L1 can be theexpression level of CHI3L1 in a prior sample obtained from the subject.This permits a direct analysis of any change in levels in thatindividual.

In some embodiments, a level of CHI3L1 can be increased relative to areference level if the level of CHI3L1 is at least 1.25× the referencelevel, e.g., at least 1.25×, at least 1.5×, at least 2×, at least 3×, atleast 4×, at least 5×, at least 6×, or greater of the reference level.In some embodiments, the expression level of CHI3L1 can be normalizedrelative to the expression level of one or more reference genes orreference proteins. In some embodiments, the expression level of CHI3L1can be normalized relative to a reference value.

In some embodiments, the expression level of no more than 20 other genesis determined. In some embodiments, the expression level of no more than10 other genes is determined.

The term “sample” or “test sample” as used herein denotes a sample takenor isolated from an organism, e.g., a urine sample from a subject.Exemplary biological samples include, but are not limited to, a biofluidsample; serum; plasma; urine; saliva; and/or tumor sample, etc. The termalso includes a mixture of the above-mentioned samples. The term “testsample” also includes untreated or pretreated (or pre-processed)biological samples. In some embodiments, a test sample can comprisecells from a subject. As used herein, the term “biofluid” refers to anyfluid obtained from a biological source and includes, but is not limitedto, blood, urine, and bodily secretions.

The test sample can be obtained by removing a sample from a subject, butcan also be accomplished by using a previously isolated sample (e.g.,isolated at a prior timepoint and isolated by the same or anotherperson). In addition, the test sample can be freshly collected or apreviously collected sample.

In some embodiments, the test sample can be an untreated test sample. Asused herein, the phrase “untreated test sample” refers to a test samplethat has not had any prior sample pre-treatment except for dilutionand/or suspension in a solution. Exemplary methods for treating a testsample include, but are not limited to, centrifugation, filtration,sonication, homogenization, heating, freezing and thawing, andcombinations thereof. In some embodiments, the test sample can be afrozen test sample, e.g., a frozen tissue. The frozen sample can bethawed before employing methods, assays and systems described herein.After thawing, a frozen sample can be centrifuged before being subjectedto methods, assays and systems described herein. In some embodiments,the test sample is a clarified test sample, for example, prepared bycentrifugation and collection of a supernatant comprising the clarifiedtest sample. In some embodiments, a test sample can be a pre-processedtest sample, for example, supernatant or filtrate resulting from atreatment selected from the group consisting of centrifugation,filtration, thawing, purification, and any combinations thereof. In someembodiments, the test sample can be treated with a chemical and/orbiological reagent. Chemical and/or biological reagents can be employedto protect and/or maintain the stability of the sample, includingbiomolecules (e.g., nucleic acid and protein) therein, duringprocessing. One exemplary reagent is a protease inhibitor, which isgenerally used to protect or maintain the stability of protein duringprocessing. The skilled artisan is well aware of methods and processesappropriate for pre-processing of biological samples required fordetermination of the level of CHI3L1 as described herein.

In some embodiments, the methods, assays, and systems described hereincan further comprise a step of obtaining a test sample from a subject.In some embodiments, the subject can be a human subject.

In some embodiments, the methods, assays, and systems described hereincan comprise creating a report based on the level of CHI3L1. In someembodiments, the report denotes raw values for CHI3L1 in the test sample(plus, optionally, the level of CHI3L1 in a reference sample) or itindicates a percentage or fold increase in CHI3L1 as compared to areference level, and/or provides a signal that the subject is at risk ofhaving, or not having cancer.

As used herein “at risk of having” refers to at least a 2-fold greaterlikelihood of having a particular condition as compared to a subjectthat did not have an elevated and/or increased level of CHI3L1, e.g., a2-fold, or 2.5-fold, or 3-fold, or 4-fold, or greater risk.

In some embodiments, the assay or method can further comprise the stepof administering an anti-CHI3L1 therapy. In some embodiments, theanti-CHI3L1 therapy comprises an isolated antibody, antibody reagent,antigen-binding portion thereof, or CAR or CAR T cell; nucleic acid;cell; or composition as described herein.

In one aspect of any of any of the embodiments, described herein is anantibody, antibody reagent, or antigen-binding portion thereof asdescribed herein conjugated to or coupled to a detectable label.

In one aspect of any of any of the embodiments, described herein is asolid support comprising an antibody, antibody reagent, antigen-bindingfragment thereof as described herein. In some embodiments of any of theaspects, the antibody, antibody reagent or antigen-binding fragmentthereof is detectably labeled. In some embodiments of any of theaspects, the solid support comprises a particle, a bead, a polymer, or asubstrate.

In one aspect of any of the embodiments, described herein is a molecularcomplex comprising at least one antibody, antibody reagent,antigen-binding fragment thereof, or CAR of as described herein bound toan CHI3L1 polypeptide.

In one aspect, described herein is a kit comprising a composition asdescribed herein, e.g., a composition comprising an antibody, antibodyreagent, antigen-binding portion thereof, or CAR as described herein. Akit is any manufacture (e.g., a package or container) comprising atleast one reagent, e.g., an antibody, the manufacture being promoted,distributed, or sold as a unit for performing the methods describedherein. In some embodiments of any of the aspects, the antibody,antibody reagent, antigen-binding fragment thereof as described hereinis immobilized on a solid support. In some embodiments of any of theaspects, the solid support comprises a particle, a bead, a polymer, or asubstrate. In some embodiments of any of the aspects, the antibody,antibody reagent or antigen-binding fragment thereof is detectablylabeled.

The kits described herein can optionally comprise additional componentsuseful for performing the methods described herein. By way of example,the kit can comprise fluids (e.g., buffers) suitable for compositioncomprising an antibody, antigen-binding portion thereof, or CAR asdescribed herein, an instructional material which describes performanceof a method as described herein, and the like. A kit can furthercomprise devices and/or reagents for delivery of the composition asdescribed herein. Additionally, the kit may comprise an instructionleaflet and/or may provide information as to the relevance of theobtained results.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment or agent) and can include, for example,a decrease by at least about 10%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

In the various embodiments described herein, it is further contemplatedthat variants (naturally occurring or otherwise), alleles, homologs,conservatively modified variants, and/or conservative substitutionvariants of any of the particular polypeptides described areencompassed. As to amino acid sequences, one of skill will recognizethat individual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters a single aminoacid or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid andretains the desired activity of the polypeptide. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles consistent with thedisclosure.

In some embodiments, the polypeptide described herein (or a nucleic acidencoding such a polypeptide) can be a functional fragment of one of theamino acid sequences described herein. As used herein, a “functionalfragment” is a fragment or segment of a peptide which retains at least50% of the wildtype reference polypeptide's activity according to theassays described below herein. A functional fragment can compriseconservative substitutions of the sequences disclosed herein.

In some embodiments, the polypeptide described herein can be a variantof a sequence described herein. In some embodiments, the variant is aconservatively modified variant. Conservative substitution variants canbe obtained by mutations of native nucleotide sequences, for example. A“variant,” as referred to herein, is a polypeptide substantiallyhomologous to a native or reference polypeptide, but which has an aminoacid sequence different from that of the native or reference polypeptidebecause of one or a plurality of deletions, insertions or substitutions.Variant polypeptide-encoding DNA sequences encompass sequences thatcomprise one or more additions, deletions, or substitutions ofnucleotides when compared to a native or reference DNA sequence, butthat encode a variant protein or fragment thereof that retains activity.A wide variety of PCR-based site-specific mutagenesis approaches areknown in the art and can be applied by the ordinarily skilled artisan.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA orcDNA. Suitable RNA can include, e.g., mRNA.

In some embodiments of any of the aspects, a polypeptide, nucleic acid,or cell as described herein can be engineered. As used herein,“engineered” refers to the aspect of having been manipulated by the handof man. For example, a polypeptide is considered to be “engineered” whenat least one aspect of the polypeptide, e.g., its sequence, has beenmanipulated by the hand of man to differ from the aspect as it exists innature. As is common practice and is understood by those in the art,progeny of an engineered cell are typically still referred to as“engineered” even though the actual manipulation was performed on aprior entity.

In some embodiments, a nucleic acid encoding a polypeptide as describedherein (e.g. an antibody or antibody reagent) is comprised by a vector.In some of the aspects described herein, a nucleic acid sequenceencoding a given polypeptide as described herein, or any module thereof,is operably linked to a vector. A vector can include, but is not limitedto, a cloning vector, an expression vector, a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

The term “isolated” or “partially purified” as used herein refers, inthe case of a nucleic acid or polypeptide, to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) that is present with the nucleic acid orpolypeptide as found in its natural source and/or that would be presentwith the nucleic acid or polypeptide when expressed by a cell, orsecreted in the case of secreted polypeptides. A chemically synthesizednucleic acid or polypeptide or one synthesized using in vitrotranscription/translation is considered “isolated.” The terms “purified”or “substantially purified” refer to an isolated nucleic acid orpolypeptide that is at least 95% by weight the subject nucleic acid orpolypeptide, including, for example, at least 96%, at least 97%, atleast 98%, at least 99% or more. In some embodiments, the antibody,antigen-binding portion thereof, or CAR described herein is isolated. Insome embodiments, the antibody, antibody reagent, antigen-bindingportion thereof, or CAR described herein is purified.

As used herein, “engineered” refers to the aspect of having beenmanipulated by the hand of man. For example, an antibody, antibodyreagent, antigen-binding portion thereof, or CAR is considered to be“engineered” when the sequence of the antibody, antibody reagent,antigen-binding portion thereof, or CAR is manipulated by the hand ofman to differ from the sequence of an antibody as it exists in nature.As is common practice and is understood by those in the art, progeny andcopies of an engineered polynucleotide and/or polypeptide are typicallystill referred to as “engineered” even though the actual manipulationwas performed on a prior entity.

As used herein, an “epitope” can be formed on a polypeptide both fromcontiguous amino acids, or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents, whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, about 9, or about 8-10 amino acids in a uniquespatial conformation. An “epitope” includes the unit of structureconventionally bound by an immunoglobulin VH/VL pair. Epitopes definethe minimum binding site for an antibody, and thus represent the targetof specificity of an antibody. In the case of a single domain antibody,an epitope represents the unit of structure bound by a variable domainin isolation. The terms “antigenic determinant” and “epitope” can alsobe used interchangeably herein. In certain embodiments, epitopedeterminants include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics.

“Avidity” is the measure of the strength of binding between anantigen-binding molecule (such as an antibody or antigen-binding portionthereof described herein) and the pertinent antigen. Avidity is relatedto both the affinity between an antigenic determinant and its antigenbinding site on the antigen-binding molecule, and the number ofpertinent binding sites present on the antigen-binding molecule.Typically, antigen-binding proteins (such as an antibody or portion ofan antibody as described herein) will bind to their cognate or specificantigen with a dissociation constant (KD of 10⁻⁵ to 10⁻¹² moles/liter orless, such as 10⁻⁷ to 10⁻¹² moles/liter or less, or 10⁻⁸ to 10⁻¹²moles/liter (i.e., with an association constant (KA) of 10⁵ to 10¹²liter/moles or more, such as 10⁷ to 10¹² liter/moles or 10⁸ to 10¹²liter/moles). Any KD value greater than 10⁻⁴ mol/liter (or any KA valuelower than 10⁴ M⁻¹) is generally considered to indicate non-specificbinding. The KD for biological interactions which are consideredmeaningful (e.g., specific) are typically in the range of 10⁻¹⁰ M (0.1nM) to 10⁻⁵ M (10000 nM). The stronger an interaction, the lower is itsKD. For example, a binding site on an antibody or portion thereofdescribed herein will bind to the desired antigen with an affinity lessthan 500 nM, such as less than 200 nM, or less than 10 nM, such as lessthan 500 pM. Specific binding of an antigen-binding protein to anantigen or antigenic determinant can be determined in any suitablemanner known per se, including, for example, Scatchard analysis and/orcompetitive binding assays, such as radioimmunoassays (RIA), enzymeimmunoassays (EIA) and sandwich competition assays, and the differentvariants thereof known per se in the art; as well as other techniques asmentioned herein.

Accordingly, as used herein, “selectively binds” or “specifically binds”refers to the ability of an peptide (e.g., an antibody, CAR or portionthereof) described herein to bind to a target, such as an antigenpresent on the cell-surface of a cancer cell, with a KD 10⁻⁵M (10000 nM)or less, e.g., 10⁻⁶ M, 10⁻⁷M, 10⁻⁸ M, 10⁻⁹M, 10⁻¹⁰ M, 10⁻¹¹M, 10⁻¹²M, orless. Specific binding can be influenced by, for example, the affinityand avidity of the polypeptide agent and the concentration ofpolypeptide agent. The person of ordinary skill in the art can determineappropriate conditions under which the polypeptide agents describedherein selectively bind the targets using any suitable methods, such astitration of a polypeptide agent in a suitable cell binding assay. Apolypeptide specifically bound to a target is not displaced by anon-similar competitor. In certain embodiments, an antibody,antigen-binding portion thereof, or CAR is said to specifically bind anantigen when it preferentially recognizes its target antigen in acomplex mixture of proteins and/or macromolecules.

In some embodiments, an antibody, antigen-binding portion thereof,and/or CAR as described herein binds to CHI3L1 with a dissociationconstant (K_(D)) of 10⁻⁵ M (10000 nM) or less, e.g., 10⁻⁶ M, 10⁻⁷ M,10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, or less. In some embodiments,an antibody, antigen-binding portion thereof, and/or CAR as describedherein binds to CHI3L1 with a dissociation constant (K_(D)) of fromabout 10⁻⁵ M to 10⁻⁶ M. In some embodiments, an antibody,antigen-binding portion thereof, and/or CAR as described herein binds toCHI3L1 with a dissociation constant (K_(D)) of from about 10⁻⁶ M to 10⁻⁷M. In some embodiments, an antibody, antigen-binding portion thereof,and/or CAR as described herein binds to CHI3L1 with a dissociationconstant (K_(D)) of from about 10⁻⁷ M to 10⁻⁸ M. In some embodiments, anantibody, antigen-binding portion thereof, and/or CAR as describedherein binds to CHI3L1 with a dissociation constant (K_(D)) of fromabout 10⁻⁸ M to 10⁻⁹ M. In some embodiments, an antibody,antigen-binding portion thereof, and/or CAR as described herein binds toCHI3L1 with a dissociation constant (K_(D)) of from about 10⁻⁹ M to10⁻¹⁰ M. In some embodiments, an antibody, antigen-binding portionthereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (K_(D)) of from about 10⁻¹⁰ M to 10⁻¹¹ M. In someembodiments, an antibody, antigen-binding portion thereof, and/or CAR asdescribed herein binds to CHI3L1 with a dissociation constant (K_(D)) offrom about 10⁻¹¹ M to 10⁻¹² M. In some embodiments, an antibody,antigen-binding portion thereof, and/or CAR as described herein binds toCHI3L1 with a dissociation constant (K_(D)) of less than 10⁻¹² M.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

One of skill in the art can readily identify a chemotherapeutic agent ofuse (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, EdwardChu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles ofCancer Therapy, Chapter 85 in Harrison's Principles of InternalMedicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era ofMolecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 inAbeloff s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

In some embodiments of any of the aspects, the disclosure describedherein does not concern a process for cloning human beings, processesfor modifying the germ line genetic identity of human beings, uses ofhuman embryos for industrial or commercial purposes or processes formodifying the genetic identity of animals which are likely to cause themsuffering without any substantial medical benefit to man or animal, andalso animals resulting from such processes.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. An antibody, antibody reagent, antigen-binding fragment thereof,    or chimaeric antigen receptor (CAR), that specifically binds an    CHI3L1 polypeptide, said antibody reagent, antigen-binding portion    thereof, or CAR comprising at least one heavy or light chain    complementarity determining region (CDR) selected from the group    consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or    -   a conservative substitution variant of one or more of (a)-(f).-   2. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of paragraph 1, which comprises heavy chain CDRs having the    amino acid sequences of SEQ ID NOs: 1-3 or a conservative    substitution variant of such amino acid sequence.-   3. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-2, which comprises light chain CDRs    having the amino acid sequences of SEQ ID NOs: 4-6 or a conservative    substitution variant of such amino acid sequence.-   4. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-3, which comprises light chain CDRs    having the amino acid sequences of SEQ ID NOs: 4-6 and heavy chain    CDRs having the amino acid sequences of SEQ ID NOs: 1-3 or a    conservative substitution variant of such amino acid sequence.-   5. An antibody, antibody reagent, antigen-binding portion thereof,    or CAR that specifically binds an CHI3L1 polypeptide, and can    compete for binding of CHI3L1 with an antibody comprising light    chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6 and    heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 1-3.-   6. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of paragraph 5, wherein the antibody, antibody reagent or    antigen-binding fragment thereof binds to the epitope of SEQ ID NO:    13.-   7. An antibody, antibody reagent, antigen-binding portion thereof,    or CAR of paragraphs 5 or 6, wherein the antibody, antibody reagent    or antigen-binding fragment thereof binds a CHI3L1 polypeptide at an    eptitope selected from SEQ ID NOs: 13-24.-   8. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-7, further comprising a conservative    substitution in a sequence not comprised by a CDR.-   9. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-8, wherein the antibody reagent or    antigen-binding fragment thereof is fully human or fully humanized.-   10. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-9, wherein the antibody reagent or    antigen-binding fragment thereof is fully humanized except for the    CDR sequences.-   11. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-10, wherein the reagent or fragment is    selected from the group consisting of:    -   an immunoglobulin molecule, a monoclonal antibody, a chimeric        antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a        Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single        domain antibody, a diabody, a multispecific antibody, a dual        specific antibody, an anti-idiotypic antibody, and a bispecific        antibody.-   12. A composition comprising the antibody, antibody reagent,    antigen-binding portion thereof, or CAR of any one of paragraphs    1-11, and a chemotherapeutic agent.-   13. The composition of paragraph 12, wherein the antibody, antibody    reagent, or antigen-binding portion thereof is conjugated to the    chemotherapeutic agent.-   14. A nucleic acid sequence encoding the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-11,    wherein at least one CDR is encoded by a nucleic acid sequence    selected from SEQ ID NOs: 7-12.-   15. A cell comprising the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-11    or the nucleic acid sequence of paragraph 14.-   16. A pharmaceutical composition comprising the antibody, antibody    reagent, antigen-binding fragment thereof, or CAR of any of    paragraphs 1-11; or the composition of any of paragraphs 12-13; or    the cell of paragraph 15, and a pharmaceutically acceptable carrier.-   17. A solid support comprising the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-11.-   18. The solid support of paragraph 17, wherein the antibody,    antibody reagent or antigen-binding fragment thereof is detectably    labeled.-   19. The solid support of any of paragraphs 17-18, wherein the solid    support comprises a particle, a bead, a polymer, or a substrate.-   20. A kit for the detection of CHI3L1 polypeptide in a sample, the    kit comprising at least a first antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-11    immobilized on a solid support and comprising a detectable label.-   21. A molecular complex comprising at least one antibody, antibody    reagent, antigen-binding fragment thereof, or CAR of any of    paragraphs 1-11 bound to a CHI3L1 polypeptide.-   22. A method of treating cancer in a subject in need thereof, the    method comprising administering the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-11;    or the composition of any of paragraphs 12-13; or the cell of    paragraph 15, to the subject.-   23. The method of paragraph 22, wherein the cancer is primary    cancer.-   24. The method of paragraph 22, wherein the cancer is malignant    cancer.-   25. The method of any of paragraphs 22-24, wherein the cancer is    selected from the group consisting of:    -   prostate cancer, colon cancer, rectal cancer, ovarian cancer,        kidney cancer, breast cancer, glioblastoma, melanoma, malignant        melanoma, and lung cancer.-   26. A method of treating asthma in a subject in need thereof, the    method comprising administering the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-11;    or the composition of any of paragraphs 12-13; or the cell of    paragraph 15, to the subject.-   27. The method of any of paragraphs 22-26 wherein the subject is a    subject determined to have an elevated level of CHI3L1.-   28. The method of paragraph 27, wherein the CHI3L1 is circulating    CHI3L1.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. An antibody, antibody reagent, antigen-binding fragment thereof,    or chimaeric antigen receptor (CAR), that specifically binds an    CHI3L1 polypeptide, said antibody reagent, antigen-binding portion    thereof, or CAR comprising at least one heavy or light chain    complementarity determining region (CDR) selected from the group    consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or    -   a conservative substitution variant of one or more of (a)-(f).-   2. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of paragraph 1, which comprises heavy chain CDRs having the    amino acid sequences of SEQ ID NOs: 1-3 or a conservative    substitution variant of such amino acid sequence.-   3. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-2, which comprises light chain CDRs    having the amino acid sequences of SEQ ID NOs: 4-6 or a conservative    substitution variant of such amino acid sequence.-   4. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-3, which comprises light chain CDRs    having the amino acid sequences of SEQ ID NOs: 4-6 and heavy chain    CDRs having the amino acid sequences of SEQ ID NOs: 1-3 or a    conservative substitution variant of such amino acid sequence.-   5. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-3, comprising a heavy chain sequence    having the amino acid sequence of SEQ ID NO: 36.-   6. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-3, comprising a light chain sequence    having the amino acid sequence of SEQ ID NO: 38.-   7. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-3, comprising a heavy chain sequence    having the amino acid sequence of SEQ ID NO: 36 and a light chain    sequence having the amino acid sequence of SEQ ID NO: 38.-   8. An antibody, antibody reagent, antigen-binding portion thereof,    or CAR that specifically binds an CHI3L1 polypeptide, and can    compete for binding of CHI3L1 with an antibody comprising light    chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6 and    heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 1-7.-   9. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of paragraph 8, wherein the antibody, antibody reagent or    antigen-binding fragment thereof binds to an epitope of SEQ ID NO:    13-24.-   10. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of paragraph 8, wherein the antibody, antibody reagent or    antigen-binding fragment thereof binds to the epitope of SEQ ID NO:    13.-   11. An antibody, antibody reagent, antigen-binding portion thereof,    or CAR that specifically binds an CHI3L1 polypeptide at an epitope    of SEQ ID NO: 13-24.-   12. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-11, further comprising a conservative    substitution in a sequence not comprised by a CDR.-   13. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-12, wherein the antibody reagent or    antigen-binding fragment thereof is fully human or fully humanized.-   14. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-13, wherein the antibody reagent or    antigen-binding fragment thereof is fully humanized except for the    CDR sequences.-   15. The antibody, antibody reagent, antigen-binding portion thereof,    or CAR of any of paragraphs 1-14, wherein the reagent or fragment is    selected from the group consisting of:    -   an immunoglobulin molecule, a monoclonal antibody, a chimeric        antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a        Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single        domain antibody, a diabody, a multispecific antibody, a dual        specific antibody, an anti-idiotypic antibody, and a bispecific        antibody.-   16. A composition comprising the antibody, antibody reagent,    antigen-binding portion thereof, or CAR of any one of paragraphs    1-15, and a chemotherapeutic agent.-   17. The composition of paragraph 16s, wherein the antibody, antibody    reagent, or antigen-binding portion thereof is conjugated to the    chemotherapeutic agent.-   18. A nucleic acid sequence encoding the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-17.-   19. A nucleic acid sequence encoding the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-15,    wherein at least one CDR is encoded by a nucleic acid sequence    selected from SEQ ID NOs: 7-12.-   20. The nucleic acid sequence of paragraph 18, comprising one or    more sequences selected from SEQ ID NOs: 37 and/or 39.-   21. A cell comprising the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-15    or the nucleic acid sequence of any of paragraphs 18-20.-   22. A pharmaceutical composition comprising the antibody, antibody    reagent, antigen-binding fragment thereof, or CAR of any of    paragraphs 1-15; or the composition of any of paragraphs 16-17; or    the cell of paragraph 21, and a pharmaceutically acceptable carrier.-   23. A solid support comprising the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-15.-   24. The solid support of paragraph 23, wherein the antibody,    antibody reagent or antigen-binding fragment thereof is detectably    labeled.-   25. The solid support of any of paragraphs 23-24, wherein the solid    support comprises a particle, a bead, a polymer, or a substrate.-   26. A kit for the detection of CHI3L1 polypeptide in a sample, the    kit comprising at least a first antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-15    immobilized on a solid support and comprising a detectable label.-   27. A molecular complex comprising at least one antibody, antibody    reagent, antigen-binding fragment thereof, or CAR of any of    paragraphs 1-15 bound to a CHI3L1 polypeptide.-   28. A method of treating cancer in a subject in need thereof, the    method comprising administering the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-15;    or the composition of any of paragraphs 16-17; or the cell of    paragraph 201, to the subject.-   29. The method of paragraph 28, wherein the cancer is primary    cancer.-   30. The method of paragraph 28, wherein the cancer is malignant    cancer.-   31. The method of any of paragraphs 28-30, wherein the cancer is    selected from the group consisting of:    -   prostate cancer, colon cancer, rectal cancer, ovarian cancer,        kidney cancer, breast cancer, glioblastoma, melanoma, malignant        melanoma, and lung cancer.-   32. A method of treating asthma in a subject in need thereof, the    method comprising administering the antibody, antibody reagent,    antigen-binding fragment thereof, or CAR of any of paragraphs 1-15;    or the composition of any of paragraphs 16-17; or the cell of    paragraph 21, to the subject.-   33. A method of treating obesity and/or preventing weight gain in a    subject in need thereof, the method comprising administering the    antibody, antibody reagent, antigen-binding fragment thereof, or CAR    of any of paragraphs 1-15; or the composition of any of paragraphs    16-17; or the cell of paragraph 21, to the subject.-   34. A method of promoting weight or fat loss in a subject in need    thereof, the method comprising administering the antibody, antibody    reagent, antigen-binding fragment thereof, or CAR of any of    paragraphs 1-15; or the composition of any of paragraphs 16-17; or    the cell of paragraph 21, to the subject.-   35. A method of treating NASH, NFALD, or metabolic syndrome in a    subject in need thereof, the method comprising administering the    antibody, antibody reagent, antigen-binding fragment thereof, or CAR    of any of paragraphs 1-15; or the composition of any of paragraphs    16-17; or the cell of paragraph 21, to the subject.-   36. The method of any of paragraphs 28-35, wherein the subject is a    subject determined to have an elevated level of CHI3L1.-   37. The method of paragraph 36, wherein the CHI3L1 is circulating    CHI3L1.

EXAMPLES Example 1: Chitinase 3-Like-1 (Chi3l1) Neutralizing Antibodiesas Therapeutics in Asthma and Lung Cancer

Members of the 18 glycosyl hydrolase (GH) gene family are dysregulatedin and play an important role in the pathogenesis of a variety ofdiseases. This is particularly striking for the chitinase-like proteincalled chitinase 3-like-1 (Chi3l1; also called Chill in mice and YKL-40in man) in asthma and lung cancer.

It has been demonstrated that the levels of circulating Chi3l1 areincreased in human asthma where they correlate with disease severity(1-4). Single nucleotide polymorphisms of Chi3l1 that correlate withincreased levels of circulating Chi3l1, asthma prevalence and poor lungfunction have been identified (2, 3, 5). In accord with the items notedabove, it was also demonstrated that null mutations of Chi3l1 markedlydecreased Th2 inflammation and eosinophil accumulation in aeroallergenmurine asthma models (6).

The levels of circulating YKL-40 are increased in many malignanciesincluding cancers of the prostate, colon, rectum, ovary, kidney, breast,glioblastomas and malignant melanoma (7-19). In these diseases, thelevels of YKL-40 frequently correlate directly with disease progressionand inversely with disease-free interval and survival (7-19). This isparticularly striking in lung cancer where the serum and tissue levelsof YKL-40 are impressively increased and correlate with adverse outcomes(20-23). To address the roles of Chi3l1 in these responses, the roles ofChi3l1 in primary and metastatic lung cancer were evaluated. Thesestudies demonstrate that (1) YKL-40 is expressed in an exaggeratedmanner in human lung cancer where it correlates inversely with survival;(2) in murine models, Chi3l1 is sequentially induced in normal peritumorand tumor tissues during the early and later stages, respectively, oflung cancer development; (3) Chi3l1 induction via a semaphorin7a-dependent mechanism plays a critical role in the generation of ametastasis permissive pulmonary microenvironment; (4) in metastaticmodels, Chi3l1 production and metastatic spread can be inhibited viaRIG-like helicase (RLH) innate immunity (24, 25). These studiesdemonstrate that Chi3l1 is induced in selective tissue compartmentsduring lung cancer initiation and progression and define the essentialrole that it plays in disease progression.

Generation and Characterization of Antibody-Based Anti-Chi3l1 BasedTherapeutics.

Because Chi3l1 is induced in patients with asthma and lung cancer andplays a critical role in the pathogenesis of murine models of bothdiseases studies were undertaken to develop and assess the efficacy ofChi3l1 neutralizing antibodies. Epitopes of Chi3l1 were selected (seeTables 1 and 2 for epitope design and selection) and monoclonalantibodies were generated.

The antibodies were then assessed for their (a) sensitivity, andspecificity of detection of Chi3l1 in human and murine systems usingdenatured and non-denatured Chi3l1; (b) binding affinity; (c) ability toneutralize the effects of rChi3l1 in cell based assays; and (d) abilityto neutralize Chi3l1 in vivo. Among the antibodies that were generatedone, called FRG, had the most exciting characteristics. It is an IgG2bKappa antibody that powerfully detects human and murine Chi3l1 underdenaturing and non-denaturing conditions with high specificity (FIGS.1A-1D). It also blocks rChi3l1 induced MAPK and AKT signaling in vitro(FIGS. 2A-2B).

To determine if FRG blocked asthma-like inflammation, the ovalbuminsensitization and challenge murine model was utilized. Wild type miceand mice in which murine Chi3l1 (Chill) was replaced by Chi3l1/YKL-40were employed. The effects on airway eosinophilic inflammation wereassessed in mice that received an IgG2b control antibody or FRG at adose of 200m/mouse. As can be seen in FIG. 3, FRG was a potent inhibitorof aeroallergen-induced eosinophilic inflammation and BAL accumulation.

To determine if FRG blocked metastatic cancer B16-F10 malignantmelanocytes were used as described previously (24, 25). These cells wereadministered by tail vein to wild type mice and pulmonary metastasiswere quantitated. Melanoma metastasis were assessed in mice thatreceived an IgG2b control antibody or FRG at a dose of 200m/mouse. Ascan be seen in FIG. 4, FRG was a potent inhibitor of melanomametastasis. Importantly, these effects were not restricted to melanomabecause similar results were seen with breast cancer cells.

Studies were next undertaken to determine if FRG ameliorated thegeneration of primary lung cancer. In these studies, primary lungcancers were induced in mice with KRAS' mutations and floxed p53mutations (26, 27) and Chi3l1/Chill expression was characterized. Inthis model a discrete mass of tumor cells and a macrophage-richinflammatory response can be seen 6 weeks after Adeno-Cre recombinasechallenge that gradually increases over time (FIG. 5). The generation ofprimary lung cancers were assessed in mice that received an IgG2bcontrol antibody or FRG at a dose of 200 μg/mouse. As can be seen inFIGS. 5A-5B, FRG was a potent inhibitor of the generation of primarylung cancers in this model.

Because of the impressive results of FRG in these in vivo models isvariable regions were sequenced. The sequences are noted in Table 3.

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TABLE 1 Parameters considered in epitope selection algorithms. SecondaryStructure Amino Acid property Loop/helix/sheet Antigenic enhancementamino acid Special region Flezibility N-terminal, C-terminal EvolutionSignal peptide Positive selection Trans-membran DiscriminationDisordered region Customer requests Solvent accessibility Proteinspecificity Blast Region specificity Query species and Mouse Others

TABLE 2 List of selected epitopes including FRG (ID Number 0) ID SEQnumber Start End Peptide ID NO  0 223 234 FRGQEDASP 13 DRF  1 304 315RGATVHRIL 14 GQQ  2 268 279 ASSETGVGA 15 PIS  3 162 173 IKEAQPGKK 16 QLL 4  62  73 SNDHIDTWE 17 WND  5 141 152 YPGRRDKQH 18 FTT  6 245 256LRLGAPASK 19 LVM  7 281 292 PGIPGRFTK 20 EAG  8 102 113 GSQRFSKIA 21 SNT 9 181 192 GKVTIDSSY 22 DIA 10  78  89 GMLNTLKNR 23 NPN 11 111 122SNTQSRRTF 24 IKS

Location of selected epitopes including FRG in human Chi3l1 (shown belowin italics; e.g., amino acids 223-234) of SEQ ID NO: 25

(SEQ ID NO: 25) MGVKASQTGFVVLVLLQCCSAYKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISNDHIDTWEWNDVTLYGMLNTLKNRNPNLKTLLSVGGWNFGSQRFSKIASNTQSRRTFIKSVPPFLRTHGFDGLDLAWLYPGRRDKQHFTTLIKEMKAEFIKEAQPGKKQLLLSAALSAGKVTIDSSYDIAKISQHLDFISIMTYDFHGAWRGTTGHHSPLFRGQEDASPDRFSNTDYAVGYMLRLGAPASKLVMGIPTFGRSFTLASSETGVGAPISGPGIPGRFTKEAGTLAYYEICDFLRGATVHRILGQQVPYATKGNQWVGYDDQESVKSKVQYLKDRQLAGAMVWALDLDDFQGSFCGQDLRFPLTNAI KDALAAT

TABLE 3 Sequences of variable complementaritydetermining regions (CDRs) of FRG antibody SEQ ID NO: Heavy CDR1GYTFTNYG  1 chain (DNA) (GGGTATACCTTCACAAACTATGGA)  7 (IgG2b) CDR2I N T Y T G E P  2 (DNA) (ATAAATACCTACACTGGAGAGCCA)  8 CDR3ARLGYGKFYVMDY  3 (DNA) (GCAAGATTGGGATATGGTAAATTCT  9 ATGTTATGGACTAC)Light CDR1 QSLVHSNGNTY  4 chain (DNA) (CAGAGCCTTGTACACAGTAATGGAA 10(IgG K) ACACCTAT) CDR2 K V S  5 (DNA) (AAAGTTTCC) 11 CDR3S Q S T H V T W T  6 (DNA) (TCTCAAAGTACACATGTTACGTGGA 12 CG)

FRG Heavy chain sequence (SEQ ID NO: 36)QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLRNEDMSTYFCARLGYGKFYVMDYWGQGTSVTVSS FRG Heavy chain nucleotide sequence(SEQ ID NO: 37) CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAATACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAGAAATGAGGACATGTCTACATATTTCTGTGCAAGATTGGGATATGGTAAATTCTATGTTATGGACTACTGGGGTCAGGGAACGTCAGTCACCGTCTCCTCA FRG Light chain sequence (SEQ ID NO: 38)DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQS THVTWTFGGGTKLEIKFRG Heavy chain nucleotide sequence (SEQ ID NO: 39)GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTACGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

Example 3

Over the course of a 14 week high fat diet (HFD), wild type miceprovided anti-Chi3L1-FRG displayed less weight gain than mice providedwith control IgG (FIG. 9). The weight gain is depicted in absoluteweight gain in FIG. 10 and the end-point weight differences are depictedin FIG. 11.

Over the course of a 12 week HFD with or without FRG antibody (200μg/twice per week), FRG administration was observed to slow weight gain(FIG. 12), reduce abdominal fat (FIG. 13) and fat pad (FIG. 14)accumulation. FRG administration also lowered serum glucose levels (FIG.15) and liver triglycerides (FIG. 16).

What is claimed herein is:
 1. An antibody, antibody reagent,antigen-binding fragment thereof, or chimaeric antigen receptor (CAR),that specifically binds an CHI3L1 polypeptide, said antibody reagent,antigen-binding portion thereof, or CAR comprising at least one heavy orlight chain complementarity determining region (CDR) selected from thegroup consisting of: (a) a light chain CDR1 having the amino acidsequence of SEQ ID NO: 4; (b) a light chain CDR2 having the amino acidsequence of SEQ ID NO: 5; (c) a light chain CDR3 having the amino acidsequence of SEQ ID NO: 6; (d) a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 1; (e) a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 2; and (f) a heavy chain CDR3 having the aminoacid sequence of SEQ ID NO:
 3. 2. The antibody, antibody reagent,antigen-binding portion thereof, or CAR of claim 1, which comprisesheavy chain CDRs having the amino acid sequences of SEQ ID NOs: 1-3. 3.The antibody, antibody reagent, antigen-binding portion thereof, or CARof claim 1, which comprises light chain CDRs having the amino acidsequences of SEQ ID NOs: 4-6.
 4. The antibody, antibody reagent,antigen-binding portion thereof, or CAR of claim 1, which compriseslight chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6 andheavy chain CDRs having the amino acid sequences of SEQ ID NOs: 1-3. 5.The antibody, antibody reagent, antigen-binding portion thereof, or CARof claim 1, comprising a heavy chain sequence having the amino acidsequence of SEQ ID NO:
 36. 6. The antibody, antibody reagent,antigen-binding portion thereof, or CAR of claim 1, comprising a lightchain sequence having the amino acid sequence of SEQ ID NO:
 38. 7. Theantibody, antibody reagent, antigen-binding portion thereof, or CAR ofclaim 1, comprising a heavy chain sequence having the amino acidsequence of SEQ ID NO: 36 and a light chain sequence having the aminoacid sequence of SEQ ID NO:
 38. 8. The antibody, antibody reagent,antigen-binding portion thereof, or CAR of claim 7, further comprising aconservative substitution in a sequence not comprised by a CDR.
 9. Theantibody, antibody reagent, antigen-binding portion thereof, or CAR ofclaim 1, wherein the antibody reagent or antigen-binding fragmentthereof is fully human or fully humanized.
 10. The antibody, antibodyreagent, antigen-binding portion thereof, or CAR of claim 1, wherein theantibody reagent or antigen-binding fragment thereof is fully humanizedexcept for the CDR sequences.
 11. The antibody, antibody reagent,antigen-binding portion thereof, or CAR of claim 1, wherein the reagentor fragment is selected from the group consisting of: an immunoglobulinmolecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, adisulfide linked Fv, a scFv, a single domain antibody, a diabody, amultispecific antibody, a dual specific antibody, an anti-idiotypicantibody, and a bispecific antibody.
 12. A nucleic acid sequenceencoding the antibody, antibody reagent, antigen-binding fragmentthereof, or CAR of claim
 1. 13. The nucleic acid sequence of claim 12,wherein at least one CDR is encoded by a nucleic acid sequence selectedfrom SEQ ID NOs: 7-12.
 14. The nucleic acid sequence of claim 12,comprising one or more sequences selected from SEQ ID NOs: 37 and 39.15. A cell comprising the antibody, antibody reagent, antigen-bindingfragment thereof, or CAR of claim
 1. 16. A pharmaceutical compositioncomprising the antibody, antibody reagent, antigen-binding fragmentthereof, or CAR of claim 1, and a pharmaceutically acceptable carrier.17. The pharmaceutical composition of claim 22, further comprising achemotherapeutic agent.
 18. The composition of claim 17, wherein theantibody, antibody reagent, or antigen-binding portion thereof isconjugated to the chemotherapeutic agent.